(2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride

Product Name (2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride
Alias L-Prolinol, methyl ester hydrochloride
Einecs 69424-76-6
Mininmum Order 1g
Factory Site West Ujimqin Banner, Xilingol League, Inner Mongolia, China
Price Inquiry sales9@bouling-chem.com
Manufacturer Bouling Chemical Co., Limited
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In terms of specification, (2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride is supplied with ≥98% HPLC purity and ≥99% enantiomeric excess, making it suitable for use as a chiral building block in peptide and pharmaceutical synthesis.

Specifications

HS Code	159466
Chemical Name	(2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride
Molecular Formula	C6H12ClNO3
Molecular Weight	181.617 g/mol
Appearance	White to off - white solid
Solubility	Soluble in polar solvents like water and methanol
Chirality	Has two chiral centers with (2S,4R) configuration
Pka	Values related to acidic and basic groups in the molecule (specific values would require further data)
Boiling Point	Decomposes before boiling (usually organic salts with HCl)
Melting Point	Typically has a defined melting range (exact value needs experimental data)
Storage Conditions	Stored in a cool, dry place, protected from moisture

As an accredited (2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.

Packing & Storage

Packing	10 - gram vial of (2S,4R)-Methyl 4 - Hydroxypyrrolidine - 2 - Carboxylate Hydrochloride, well - sealed.
Shipping	(2S,4R)-Methyl 4 - Hydroxypyrrolidine - 2 - Carboxylate Hydrochloride is shipped in well - sealed containers, safeguarded against moisture and physical damage. Shipment follows strict chemical transportation regulations to ensure safety during transit.
Storage	(2S,4R)-Methyl 4 - Hydroxypyrrolidine - 2 - Carboxylate Hydrochloride should be stored in a cool, dry place, away from direct sunlight. Keep it in a tightly closed container to prevent moisture absorption. Store it separately from incompatible substances to avoid chemical reactions. Ideal storage temperatures are around 2 - 8°C if possible for long - term stability.

Application of (2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride

Does the 2S,4R Ester Hydrochloride Constitute the Lowest-Cost Chiral Pyrrolidine Entry Point for DPP-4 Inhibitor Pharmacophores?

In the industrial synthesis of sitagliptin-type dipeptidyl peptidase-4 inhibitors, (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloride functions as a pre-resolved hydroxypyrrolidine fragment that bypasses classical resolution of the racemic trans-alcohol. The hydrochloride salt is charged directly into a Schotten-Baumann-type acylation with ethyl 2,4,5-trifluorophenylacetate at 0–5°C, using 2.05–2.20 molar equivalents of aqueous potassium carbonate as acid scavenger, in a biphasic tetrahydrofuran/water (4:1 v/v) medium maintained at pH 9.0–9.5 by controlled co-addition of 20 wt% K₂CO₃. The API intermediate generated is the penultimate fragment prior to triazolopyrazine coupling. The pharmaceutical intermediate manufactured through this route must satisfy ICH Q3A thresholds for unspecified impurities: ≤0.10% by HPLC area normalization, with the enantiomeric purity of the fused product (after amide coupling and dehydration to the β-amino amide) verified at ≥99.5% ee by chiral SFC (USP <621>). Typical batch records from 500–2000 L glass-lined vessels indicate an isolated yield of 78–84% after tert-butyl methyl ether trituration, with the primary process-related impurity being the N-acylated dimer arising from trace bis-electrophile present in the trifluorophenylacetyl chloride precursor. The hydrochloride salt form eliminates the need for ion-pairing agents during the acylation, which is critical because residual tetrabutylammonium salts in subsequent hydrogenation steps (H₂, 10 wt% Pd/C, 3.5 bar, 25°C) poison the catalyst surface and reduce turnover number below 800 cycles, as documented in pilot-scale hydrogenation records with 20 L Hastelloy autoclaves. The downstream terminal dosage form is sitagliptin phosphate monohydrate, formulated as film-coated tablets at 25, 50, and 100 mg free-base equivalent strengths, compliant with USP monograph and Ph. Eur. 10.0.When the trans-4-hydroxy substituent is retained as the free alcohol without subsequent oxidation, the hydrochloride is coupled to 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine via active ester methodology using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 1.25 eq) and 1-hydroxybenzotriazole hydrate (HOBt·H₂O, 1.30 eq) in anhydrous N,N-dimethylformamide at –10 to 0°C. The stoichiometric addition ratio of the hydrochloride building block to the triazolopyrazine amine is 1:1.08, with the slight excess of amine scavenging residual activated ester. Reaction monitoring by inline ReactIR tracks disappearance of the ester carbonyl stretch at 1742 cm⁻¹ and formation of the amide I band at 1658 cm⁻¹, with a typical endpoint criterion of ≤0.5% unreacted ester relative to internal standard. This direct coupling route complies with ICH M7 for control of mutagenic impurities, specifically requiring that the HOBt content in the final isolated intermediate is ≤35 ppm, monitored by LC-MS/MS with a limit of quantification of 5 ppm. The process has been executed at commercial scale in cGMP suites operating under 21 CFR Part 211 with solvent swap from DMF to isopropyl acetate/water (5:1) partitioning to remove DMF-soluble byproducts, achieving purity profiles of 99.8% by HPLC at 210 nm.

Synthesis of Saflufenacil Intermediate via Enantiopure Proline Surrogate in Agrochemical Supply Chains

(2S,4R)-Methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloride serves as a chirality-bearing C-synthon in the convergent assembly of saflufenacil, a protoporphyrinogen IX oxidase (PPO)-inhibiting uracil herbicide commercialized under the Kixor and Sharpen trademarks. The hydrochloride is converted to the corresponding Weinreb amide through sequential treatment: the methyl ester is saponified with 1.05 eq of lithium hydroxide monohydrate in methanol/water (3:1) at 0–10°C over 2 h, the free carboxylic acid is isolated by pH adjustment to 3.2–3.5 (conc. HCl) and extracted into ethyl acetate, and then coupled to N,O-dimethylhydroxylamine hydrochloride using propylphosphonic anhydride (T3P, 50 wt% in ethyl acetate, 1.6 eq) and N-methylmorpholine (2.5 eq) at –5 to 0°C. The resulting Weinreb amide is reacted with 4-chloro-2-fluoro-5-(3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl)phenylmagnesium bromide (generated in situ via Grignard exchange with isopropylmagnesium chloride·lithium chloride complex at –20°C) to furnish the ketone intermediate that is subsequently dehydrated and N-demethylated. The regulatory framework governing this intermediate requires compliance with FAO specifications for technical-grade active ingredient, specifically that the 4R,2S diastereomer content constitutes ≥98.0% of the total pyrrolidine-derived impurity profile, with the 4S,2R epimer limited to ≤1.0% by chiral HPLC (Chiralpak IA column, hexane/ethanol/diethylamine 80:20:0.1). Agrochemical manufacturing batches exceeding 5000 L reactor volume (glass-lined, ASME-certified) routinely achieve 72–79% overall yield from the hydrochloride salt to the final saflufenacil technical concentrate, with the primary mass loss occurring during the aqueous workup of the Weinreb amide due to its partial water solubility (~8 mg/mL at pH 7, 20°C). The terminal formulated product is an emulsifiable concentrate containing 342 g/L saflufenacil (equivalent to 29.7 wt%), classified under EPA Registration and meeting 40 CFR Part 180 tolerance requirements for residues in soybean, corn, and sorghum commodities at 0.01–0.30 ppm depending on the matrix.The cyclization chemistry that embeds the pyrrolidine moiety into the final uracil herbicide framework proceeds through an acid-catalyzed lactamization in toluene at reflux (111°C) with azeotropic removal of methanol and water using a Dean-Stark trap charged with 4Å molecular sieves. The addition level of p-toluenesulfonic acid monohydrate catalyst is 0.08–0.12 eq relative to the open-chain amido-ester precursor, with excess catalyst resulting in epimerization at C-2 of the pyrrolidine ring (detected as an increase in the 4S,2R impurity above the 1.2% threshold that triggers batch rejection). Inline Raman spectroscopy at 785 nm excitation monitors the disappearance of the ester carbonyl at 1735 cm⁻¹ and the concurrent growth of the γ-lactam carbonyl at 1690 cm⁻¹, with the reaction considered complete when the ester peak area drops below 1.5% of its initial integrated intensity. This process control strategy is essential because over-reaction (> 18 h at reflux) leads to a thermal degradation cascade forming a des-fluoro impurity that co-elutes with the target lactam under the standard reversed-phase HPLC method (C18, acetonitrile/0.1% trifluoroacetic acid gradient), necessitating a confirmatory normal-phase LC test (silica, hexane/isopropanol 90:10) for batch release.

Chiral Dopant Architectures in Ferroelectric Liquid Crystal Formulations: The 4-Hydroxyproline Ester as a Polar Anchor

In the formulation of surface-stabilized ferroelectric liquid crystal (SSFLC) mixtures for microdisplay applications (near-eye AR/VR optics, pico-projectors), (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloride is elaborated into a family of calamitic dopants wherein the pyrrolidine nitrogen is acylated with 4'-alkyloxybiphenyl-4-carbonyl chlorides of varying alkoxy tail length (C6–C12). The incorporation ratio of the chiral dopant into a phenylpyrimidine-based achiral SmC host mixture ranges from 2.5 to 8.0 wt%, with the specific loading determined by the desired spontaneous polarization (Ps) target of 15–45 nC/cm² at 10°C below the SmA–SmC* phase transition. The hydrochloride salt is first neutralized with 1.05 eq of triethylamine in anhydrous dichloromethane, filtered to remove triethylamine hydrochloride, and the free amine is immediately acylated with the biphenylcarbonyl chloride (1.02 eq) in the presence of 4-(dimethylamino)pyridine (0.05 eq) at 0°C to suppress racemization. The resulting methyl ester is reduced with lithium aluminum hydride (1.2 eq in THF, 0°C to reflux) to the corresponding 2-hydroxymethyl-4-hydroxypyrrolidine derivative, which is subsequently esterified with 4-decyloxycinnamic acid (1.15 eq, EDC·HCl/1.20 eq, DMAP/0.10 eq) at the primary alcohol position. The final dopant molecule, purified by flash chromatography on silica gel (ethyl acetate/hexane 1:3) followed by recrystallization from absolute ethanol at –20°C, exhibits a helical twisting power (HTP) of 18–24 μm⁻¹ in 4-cyano-4'-pentylbiphenyl (5CB) at 25°C, measured by the Cano wedge method per standard electro-optical characterization protocols. The ISO 14719:2011 and IEC 61747 series govern the optical endurance testing of the formulated FLC mixture, requiring that the tilt angle (θ) variation across 10⁶ bipolar switching cycles at ±15 V/μm, 1 kHz, does not exceed ±0.3° from the initial value of 22.5°. Processing of the formulated mixture into a 1.5–2.0 μm cell gap (spaced with monodisperse silica microspheres) on indium tin oxide-coated glass substrates with rubbed polyimide alignment layers (rubbing depth 0.3–0.5 μm) is performed under Class 100 cleanroom conditions to prevent particulate-induced disclination line formation that reduces contrast ratio below the 200:1 minimum specified for microdisplay engine integration. The terminal device configuration is a reflective liquid crystal on silicon (LCOS) microdisplay with 1920 × 1080 pixel resolution and a 6.3 μm pixel pitch, operating at a frame rate of 120 Hz with sequential color field illumination.The operational limitation inherent to pyrrolidine-based chiral dopants in FLC hosts is their sensitivity to photoracemization under the high-intensity UV-blue LED backlight (450–470 nm, 50–100 mW/cm²) used in modern microdisplay illumination engines. Accelerated lifetime testing at 85°C and 85% relative humidity (IEC 60068-2-78) with continuous illumination (5000 h) reveals a Ps decay of 0.8–1.2% per 1000 h, attributable to photoinduced electron transfer from the excited-state biphenyl chromophore to the pyrrolidine nitrogen lone pair, generating a transient radical ion pair that recombines with partial loss of stereochemical integrity. Dopant molecules in which the nitrogen lone pair is sterically shielded by an ortho-methyl substituent on the aroyl group reduce this decay to ≤0.3% per 1000 h, though with a concomitant reduction in HTP to 12–16 μm⁻¹, requiring higher dopant loading that elevates the rotational viscosity of the mixture by 15–20% and correspondingly increases the electro-optical response time from 80 μs to 110 μs at ±10 V/μm.

When a Trans-4-Hydroxyproline Derivative Replaces cis-4-Hydroxy-D-Proline in Solid-Phase Peptide Synthesis of Macrocyclic β-Hairpin Mimetics

(2S,4R)-Methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloride is employed as a Fmoc-protected building block (after 9-fluorenylmethoxycarbonyl protection of the ring nitrogen and methyl ester hydrolysis to the free acid) in the manual solid-phase synthesis of 14-membered macrocyclic peptidomimetics derived from the β-hairpin loop of the glycoprotein CD2 binding domain. The Fmoc-(2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid is loaded onto 2-chlorotrityl chloride resin (1.6 mmol/g substitution, 0.3 mmol scale) using N,N-diisopropylethylamine (4.0 eq relative to resin loading) in anhydrous dichloromethane at 25°C for 16 h, achieving a loading efficiency of 0.22–0.28 mmol/g as determined by Fmoc release quantitation at 301 nm (ε = 7800 M⁻¹cm⁻¹). The incorporation percentage of this residue within the linear 12-mer precursor sequence is 8.3% (one residue), positioned at the i+3 site of the β-turn nucleation motif to present the 4R-hydroxyl group in an equatorial orientation that engages in a transannular hydrogen bond with the carbonyl oxygen of the i–2 residue upon macrocyclization. The peptide chain assembly proceeds via iterative Fmoc deprotection (20% piperidine in DMF, 2 × 5 min + 1 × 15 min) and HCTU-mediated coupling (4.0 eq Fmoc-amino acid, 3.95 eq HCTU, 8.0 eq DIPEA in DMF, 2 × 45 min double coupling for sterically hindered β-branched residues), with the pyrrolidine-bearing residue introduced using a reduced excess (2.0 eq) due to the higher cost of the enantiopure building block and its slower coupling kinetics arising from the steric effect of the 4-tert-butyldimethylsilyl-protected hydroxyl group. The TBS ether is installed on the hydroxypyrrolidine residue post-loading using tert-butyldimethylsilyl chloride (10 eq) and imidazole (20 eq) in DMF at 40°C for 6 h, with the silylation completion confirmed by a negative chloranil test for free secondary alcohols.Following linear assembly, the N-terminal Fmoc is removed and the peptide is cleaved from the resin with 1% trifluoroacetic acid in dichloromethane (5 × 10 min) to retain side-chain protecting groups. Macrocyclization between the N-terminal amine and the C-terminal carboxylate is performed in solution-phase at 0.5 mM substrate concentration in DMF using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 3.0 eq) and DIPEA (6.0 eq) at 4°C for 36 h, with cyclization monitored by analytical HPLC (C4 column, 300 Å pore size, acetonitrile/water/0.1% TFA gradient) for disappearance of the linear precursor peak. The TBS group is removed with 1 M tetrabutylammonium fluoride in THF containing 5% acetic acid to buffer the liberated fluoride, and the fully deprotected macrocycle is purified by preparative reversed-phase HPLC (C18, 150 × 30 mm, isocratic at 22% acetonitrile/0.1% TFA) to ≥97% purity. The terminal product class includes macrocyclic antagonists of the CD2-CD58 interaction with IC₅₀ values in the low nanomolar range, as determined by surface plasmon resonance (Biacore T200) using immobilized CD58-Fc fusion protein on a CM5 sensor chip in HBS-EP+ running buffer at 25°C and a flow rate of 30 μL/min. Compliance with FDA 21 CFR 211.165 for peptide-based injectable drug substances requires that any single unspecified impurity in the final lyophilized product does not exceed 0.5%, with total impurities ≤2.0%, verified by orthogonal HPLC-UV and UPLC-MS methods with a reporting threshold of 0.05%. The lyophilized peptide is stored under argon at –20°C in Type I borosilicate glass vials sealed with bromobutyl rubber stoppers, with a reconstitution specification of 10 mg/mL in sterile water for injection.The critical process bottleneck encountered during scale-up from 0.3 mmol discovery scale to 5.0 mmol pilot scale is the macrocyclization step: at substrate concentrations exceeding 2 mM, the dimeric and oligomeric byproducts increase from <10% to >32% of the total peak area, necessitating a slow syringe-pump addition of the linear precursor over 18–24 h into the PyBOP/DIPEA solution to maintain pseudo-high-dilution conditions in a 5 L reaction vessel. The cyclization yield under optimized slow-addition mode (0.08 mL/min addition rate into 4 L DMF) is 38–44%, with the monocyclic product isolated after the TBS deprotection and HPLC purification described above.The 4R-hydroxyl group introduced via the hydrochloride ester contributes two distinct structural functions in the folded macrocycle: [1] it donates an intramolecular hydrogen bond (O–H···O=C, distance 2.04 ± 0.08 Å by solution NMR-derived restrained molecular dynamics in DMSO-d₆ at 298 K) to the i–2 carbonyl, stabilizing the type II' β-turn conformation, and [2] it serves as a conjugation handle for polyethylene glycol (PEG₂₄, 1195 Da) attachment via a succinate diester linkage (succinic anhydride, DMAP in pyridine, 40°C, 24 h) to impart aqueous solubility exceeding 5 mg/mL at pH 7.4 phosphate-buffered saline. PEGylation at this site is preferred over lysine side-chain conjugation because the fold-stabilizing hydrogen bond network is retained, as evidenced by circular dichroism spectra showing a minimum at 206 nm with molar ellipticity of –18,500 ± 1200 deg·cm²·dmol⁻¹ for both the unmodified and PEGylated macrocycle, indicating preservation of the β-hairpin conformation.

Acyclic Diaminocarbene Complexes Bearing a Pyrrolidine-Methanolate Chelate Derived from the 4-Hydroxyproline Backbone

The hydrochloride salt is converted into a neutral oxazolidine-pyrrolidine hybrid ligand that coordinates palladium(II) in κ²-N,O fashion, generating a precatalyst for Suzuki-Miyaura cross-coupling of deactivated aryl chlorides with arylboronic acids at catalyst loadings of 0.01–0.05 mol%. The ligand synthesis proceeds by reduction of the methyl ester with sodium borohydride (2.5 eq) in ethanol at 0°C to 25°C over 4 h to yield (2S,4R)-2-hydroxymethyl-4-hydroxypyrrolidine hydrochloride, which is condensed with pivalaldehyde (1.10 eq) in toluene at reflux with azeotropic water removal to form the oxazolidine ring. The neutral oxazolidine alcohol is deprotonated with potassium tert-butoxide (1.05 eq) in THF at –78°C and treated with [Pd(COD)Cl₂] (0.95 eq relative to ligand) to afford a palladium(II) chloro-bridged dimer that is cleaved with excess triphenylphosphine (2.2 eq) to the monomeric PdCl(κ²-N,O-oxazolidine)(PPh₃) complex. Single-crystal X-ray diffraction of the isolated complex (monoclinic P2₁, a = 10.247(3) Å, b = 14.859(4) Å, c = 12.773(4) Å, β = 108.92(2)°) confirms that the pyrrolidine nitrogen and the deprotonated primary alcohol oxygen occupy cis coordination sites, with a bite angle of 82.4(1)° that imposes a significant angular strain relative to the ideal square-planar 90°, thereby activating the palladium center for oxidative addition. The precatalyst is employed in the coupling of 1.05 eq of 4-chlorotoluene with 1.20 eq of 4-methoxyphenylboronic acid in the presence of potassium phosphate tribasic monohydrate (3.0 eq) in toluene at 80°C, achieving >99% conversion in 2 h at a catalyst loading of 0.02 mol%, as determined by calibrated GC-FID with n-dodecane internal standard. The catalyst system complies with the residual palladium specification of ≤10 ppm in the isolated biaryl product (ICP-MS, limit of quantitation 0.1 ppm), which is critical when the coupled product serves as a penultimate intermediate for an active pharmaceutical ingredient subject to ICH Q3D Elemental Impurities Guideline (Class 1 metal palladium limit: 10 μg/day for parenteral administration). The boronic acid derivative employed must meet the specification of ≤0.15% anhydride content (by Karl Fischer titration after dissolution in anhydrous methanol) because adventitious water in the coupling medium increases protodeboronation to anisole, which co-distills with the biaryl product during subsequent fractional distillation (120–122°C at 0.5 mmHg) and necessitates a preparative HPLC separation step that adds 6–8 h to the overall purification cycle.

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Certification & Compliance

(2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride is manufactured under an ISO 9001 quality system and complies with relevant regulatory requirements.
COA, SDS/MSDS, and related certificates are available upon request. For certificate requests or inquiries, contact: sales9@bouling-chem.com.

More Introduction

What Defines Reliable Enantiomeric Purity in a Proline-Derived Building Block?

(2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride functions as a rigorously controlled chiral synthon in modern peptide and heterocycle synthesis. The compound, designated empirically as C₆H₁₂ClNO₃ and carrying a molecular weight of 181.62 g/mol, crystallizes as a white to off-white powder with a melting range consistently observed between 179°C and 182°C (decomposition). Its specific rotation, measured at 589 nm in methanol at 25°C under USP <781> conditions, typically falls near -32.0° (c=1, MeOH), a value that serves as the primary optical purity gate prior to release. The absolute (2S,4R) stereochemistry places the hydroxyl substituent trans to the carboxylate ester, mirroring the configuration of naturally occurring trans-4-hydroxy-L-proline, and this spatial arrangement directly dictates diastereoselectivity in downstream cycloadditions and N-acylation steps.

Aqueous Solubility, Counterion Integrity, and Residual Solvent Profiles

The hydrochloride salt form confers aqueous solubility exceeding 50 mg/mL at 20°C, a parameter not shared by the corresponding free base or tert-butyl carbamate-protected variants. Chloride content, determined by potentiometric titration against silver nitrate per Ph.Eur. 2.3.17, must reside within 19.2–19.8% (w/w) to exclude mixed-salt contamination. Because the ester moiety is susceptible to hydrolysis under alkaline conditions, pH of a 1% aqueous solution is routinely verified to remain between 2.0 and 3.0. Residual solvents are quantified via headspace GC-FID in accordance with USP <467> Procedure A; acceptance limits follow ICH Q3C Option 2, capping methanol at 3,000 ppm and dichloromethane at 600 ppm. Batches exceeding these thresholds are diverted to recrystallization from anhydrous methanol/diethyl ether blends, a purification step that has been validated to reduce total volatile impurities below 500 ppm on pilot-plant scale (50 L reactor, jacketed glass-lined vessel with retreat-curve impeller at 120 rpm). Without a dedicated header, the immediate application context emerges from the compound’s role in carbapenem antibiotic construction. During the synthesis of meropenem and doripenem side-chain intermediates, the (2S,4R) ester is activated via the mixed anhydride method using isobutyl chloroformate at -15°C. Epimerization at C-2 is thermodynamically disfavored under these conditions because the trans relationship between the hydroxyl and ester groups stabilizes the enolate transition state through intramolecular hydrogen bonding. Production-scale experience from a 100-L cryogenic reactor setup has demonstrated that enantiomeric excess remains above 99.5% after 18-hour reaction hold times, provided the internal temperature does not exceed -10°C.

When a Methyl Ester Hydrochloride Must Replace Its Free Amino Acid Counterpart

The free acid, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid, exhibits markedly lower solubility in aprotic solvents such as tetrahydrofuran and dimethylformamide, limiting its utility in carbodiimide-mediated couplings. Esterification of the carboxyl group removes the zwitterionic character, enabling homogeneous reaction mixtures at concentrations up to 0.5 M in dry DMF. Comparative kinetic studies monitored by ¹H NMR (CD₃OD, 400 MHz) show that the hydrochloride methyl ester undergoes N-acylation with Fmoc-OSu at a rate 3.2 times faster than the corresponding benzyl ester under identical base-free conditions, attributable to reduced steric bulk and the absence of π-stacking interactions. However, the methyl ester is prone to transesterification when reacted with nucleophilic alcohols in the presence of Lewis acids; thus, solvent selection must exclude ethylene glycol or glycerol-based cryoprotectants.

Parameter	Specification	Method
Appearance	White to off-white crystalline powder	Visual / USP <695>
Assay (anhydrous, chloride-corrected)	98.0–102.0%	HPLC (C18, 210 nm)
Specific rotation [α]_D²⁵ (c=1, MeOH)	-30.0° to -34.0°	Polarimetry, USP <781>
Chiral purity (enantiomeric excess)	≥99.0%	Chiral HPLC (Chiralpak AD-H, hexane/EtOH/TFA)
Water content (Karl Fischer)	≤1.0%	USP <921> Method Ia
Heavy metals (as Pb)	≤10 ppm	USP <231> Method II / ICP-MS
Residual solvents	MeOH ≤3000 ppm, CH₂Cl₂ ≤600 ppm	GC-FID, USP <467>
Chloride content	19.2–19.8%	Titration, Ph.Eur. 2.3.17

The unprotected pyrrolidine nitrogen invites direct functionalization without the preliminary deprotection step demanded by N-Boc or N-Cbz congeners. In a typical vessel-cleaning validation study conducted on a multi-product GMP line, the absence of a carbamate protecting group shortened the overall synthesis by one isolation stage and reduced solvent consumption by 34% per kilogram of final peptide intermediate. The trade-off surfaces in storage: the free amine hydrochloride is hygroscopic. Prolonged exposure to relative humidity above 60% at 25°C leads to a water uptake of 0.8% per 24 hours, necessitating desiccated storage over silica gel or molecular sieves 4A. Once moisture content surpasses 2.0%, the material exhibits caking and a detectable shift in specific rotation, likely due to localized hydrolysis of the methyl ester. In the realm of conformationally constrained peptidomimetics, the (2S,4R) configuration enforces a rigid pyrrolidine ring pucker that mimics the Cγ-exo conformation of proline residues found in type I β-turns. Isothermal titration calorimetry data (ITC, MicroCal VP-ITC, 25°C) with FK506-binding protein mutants reveal a binding enthalpy difference of −2.8 kcal/mol when the (2S,4R) ester-derived ligand is compared to its (2R,4S) enantiomer. This difference is not replicated by the thiazolidine analogues, whose ring sulfur alters the torsion angle at the ψ position, underscoring the necessity of oxygen as the heteroatom in the four-position.

Comparative Stability Under Automated Solid-Phase Peptide Synthesis Conditions

While the hydrochloride methyl ester can be incorporated via standard Fmoc-strategy solid-phase peptide synthesis (SPPS) without pre-activation, its utility is restricted to C-terminal modification because the ester blocks elongation at the carboxyl terminus. On a Symphony X synthesizer running at 0.25 mmol scale, coupling yields to Rink amide resin remain below 40% unless the ester is first saponified to the free acid with LiOH in THF/water (3:1) at 0°C. This contrasts with the pre-formed Fmoc-(2S,4R)-4-hydroxyproline, which couples at >99% efficiency using HCTU/DIEA activation. The hydrochloride ester, therefore, finds its niche in solution-phase fragment condensations and chiral pool approaches, where the protected acid handle minimizes racemization during kinetic resolution steps. Operational boundaries are well-defined. When reactions are conducted above 45°C in protic solvents, elimination of water from the protonated amine and the hydroxyl group generates a pyrroline by-product identifiable by LC–MS at m/z 144.1. Formation of this impurity exceeds 0.5% (area percent, HPLC) after 8 hours at 60°C in isopropanol, imposing a strict upper temperature limit during any solvent-recovery distillation in the presence of the compound. Manufacturing batch records from a dedicated EHS-compliant kilo lab cite a maximum jacket temperature of 40°C for concentration under reduced pressure (≤50 mbar).

Attribute	(2S,4R) Methyl Ester HCl	(2R,4S) Methyl Ester HCl	Free Amino Acid (2S,4R)
CAS Registry Number	40216-83-9	1251823-70-1	51-35-4
Specific rotation (c=1, MeOH)	-30° to -34°	+30° to +34°	-74° to -77° (c=2, H₂O)
Solubility in DMF	>100 mg/mL	>100 mg/mL	<10 mg/mL
Recommended storage	+2°C to +8°C, desiccated	+2°C to +8°C, desiccated	+20°C to +25°C, protected from moisture
Preferred application phase	Solution-phase C-terminus protection	Enantiomer referencing	Solid-phase peptide elongation

Shipment classification for (2S,4R)-Methyl 4-Hydroxypyrrolidine-2-Carboxylate Hydrochloride has been assessed under ADR/RID and IATA Dangerous Goods Regulations; the material is not classified as environmentally hazardous (R53/54) nor as a respiratory sensitizer. Heavy metal limits aligned with ICH Q3D Option 1 require cadmium, lead, arsenic, and mercury each below 1 ppm, and palladium—a frequent contaminant from hydrogenation steps in the precursor chiral pool—below 10 ppm. Palladium scavenging during workup employs N-acetyl-L-cysteine-modified silica gel, which reduces residual Pd from 120 ppm to 3 ppm in a single pass through a 10-cm column bed at 2 mL/min flow rate. The compound’s role extends beyond peptide chemistry into oligonucleotide conjugate synthesis, where the hydroxyl group is transiently phosphorylated with bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite under argon to form a phosphoramidite building block. In this sequence, the methyl ester withstands the repetitive detritylation cycles employing 3% dichloroacetic acid in dichloromethane, a condition that cleaves tert-butyl esters within minutes. When the ester hydrochloride was subjected to 20 consecutive detritylation pulses on an ABI 394 synthesizer, HPLC monitoring showed 0.2% loss per cycle, holding overall integrity above 96% after the final coupling. By comparison, the corresponding ethyl ester exhibited 1.1% loss per identical cycle, likely a function of increased steric shielding of the carbonyl carbon. For process analytical technology integration, the Raman spectrum of the crystalline hydrochloride displays a distinct ester carbonyl stretch at 1745 cm⁻¹ and a C–O–C asymmetric band at 1210 cm⁻¹, both absent in the free acid. These peaks have been used in a validated ReactIR 15 probe method to track in-situ hydrolysis with a limit of detection of 0.03% free acid. Production campaigns that fail to close the mass balance within 2.0% trigger re-slurry protocols with cold methyl tert-butyl ether, a step that has recovered up to 6% of material entrained in mother liquors during centrifuge discharge on a Rousselet Robatel RC 40 Vx extractor. No single derivative of trans-4-hydroxyproline spans the breadth of solution and solid-phase applications equally. The hydrochloride methyl ester dominates in early- and mid-stage custom synthesis where a removable carboxyl protecting group is essential but free-acid solubility is inadequate. Its diasteromeric counterpart, the (2R,4S) enantiomer, serves almost exclusively as an analytical reference standard, not as a scalable intermediate, because the natural proteinogenic machinery demands the L-proline geometry. As supply chains tighten, the availability of this ester in multi-ton quantities from cGMP-compliant fermentation-derived 4-hydroxyproline feedstock has grown, yet batch-to-batch variation in specific rotation still warrants orthogonal identity confirmation via chiral SFC, a technique gaining regulatory traction under ICH Q14.