(2S,4S)-1-(Tert-Butoxycarbonyl)-4-Methylpyrrolidine-2-Carboxylic Acid
|
HS Code |
732529 |
| Chemical Formula | C12H21NO4 |
| Molecular Weight | 243.30 |
| Appearance | Solid (usually white or off - white) |
| Physical State At Room Temp | Solid |
| Melting Point | Typically in a certain range (specific value depends on purity) |
| Solubility In Water | Poorly soluble |
| Solubility In Organic Solvents | Soluble in some organic solvents like dichloromethane |
| Chirality | Chiral, with (2S,4S) configuration |
| Functional Groups | Tert - butoxycarbonyl group, carboxylic acid group, pyrrolidine ring |
As an accredited (2S,4S)-1-(Tert-Butoxycarbonyl)-4-Methylpyrrolidine-2-Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of (2S,4S)-1-(Tert - Butoxycarbonyl)-4 - Methylpyrrolidine - 2 - Carboxylic Acid in sealed, labeled container. |
| Shipping | (2S,4S)-1-(Tert -Butoxycarbonyl)-4 -Methylpyrrolidine-2 -Carboxylic Acid is shipped in well -sealed containers, compliant with chemical transportation regulations. Shipment ensures protection from moisture, heat, and physical damage during transit. |
| Storage | (2S,4S)-1-(Tert - Butoxycarbonyl)-4 - Methylpyrrolidine - 2 - Carboxylic Acid should be stored in a cool, dry place, away from heat sources and direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could potentially lead to degradation. Store in a well - ventilated area, away from incompatible substances. |
A dedicated 5000-litre campaign manufacturing train operated under cGMP conditions was configured for the industrial solid-phase peptide synthesis of a 14-residue macrocyclic peptide containing a (4S)-methyl-L-proline unit at position 7. The title compound was pre-dissolved in anhydrous N,N-dimethylformamide (DMF, water content ≤ 50 ppm by Karl Fischer) and activated in situ with 3.0 equivalents of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 6.0 equivalents of N,N-diisopropylethylamine (DIEA) at a jacket setpoint of 0 ± 2 °C. The activated ester was coupled to the resin-bound heptapeptide intermediate on a 300-kg-scale 2-chlorotrityl chloride polystyrene support with a substitution level of 0.85 mmol/g. Coupling progression was tracked by the Kaiser test until a negative result was obtained, confirming >99.7% conversion; a second recoupling cycle employing 1.5 equivalents of activated amino acid was mandated whenever a trace blue colour persisted beyond 45 minutes. Following linear assembly, global side-chain deprotection and cleavage from the resin were achieved with a trifluoroacetic acid (TFA)–triisopropylsilane–water (95:2.5:2.5 v/v/v) cocktail, and the crude peptide was precipitated with chilled methyl tert-butyl ether at −20 °C. Residual TFA counter-ions were removed by ion-exchange chromatography on a Dowex 1×8 chloride form column, reducing TFA content to ≤ 100 ppm as verified by ion chromatography per USP <1065>. The final cyclic peptide was purified by preparative reversed-phase HPLC on a C18 stationary phase with a water–acetonitrile gradient containing 0.1% TFA, yielding an active pharmaceutical ingredient that met ICH Q3C residual solvent limits for DMF (≤ 880 ppm), dichloromethane (≤ 600 ppm), and acetonitrile (≤ 410 ppm). The intact precursor integrity was monitored by liquid chromatography–high-resolution mass spectrometry, demanding an observed monoisotopic mass within 5 ppm of the theoretical value. The isolated peptide lyophilizate, incorporating the conformationally restrained (4S)-methylpyrrolidine moiety, was stored at −20 ± 5 °C under argon and used in a late-stage clinical programme targeting a G protein-coupled receptor with picomolar binding affinity.Residual Palladium Scavenging after Hydrogenolytic Debenzylation of a Proline-Containing Tripeptide IntermediateWhen the downstream route requires liberating the pyrrolidine nitrogen without disturbing a benzyl ester present on an adjacent residue, catalytic hydrogenation over 10% palladium on carbon (dry basis, Type 487, Johnson Matthey) is employed to selectively cleave the Cbz protecting group while preserving the Boc-protected title compound in solution. The hydrogenation is run in a 3000-litre Hastelloy C-276 autoclave at 3.0 barg hydrogen pressure and 25 ± 2 °C with tetrahydrofuran–methanol (4:1 v/v) as the solvent system; agitation is maintained at 800 rpm using a gas-entrainment impeller. Upon > 99% conversion confirmed by UPLC area-percent at 210 nm, the catalyst is removed by inline filtration through a 0.5 µm sintered metal cartridge followed by a 0.2 µm polytetrafluoroethylene membrane. The critical quality attribute at this stage is residual palladium content, which must not exceed 10 µg/g in the isolated tripeptide intermediate per ICH Q3D Elemental Impurities Guideline for parenteral administration. Consequently, a post-filtration treatment with 3% w/w activated carbon (Norit SX Ultra, steam-activated) and 0.5% w/w trimercapto-s-triazine-functionalized silica scavenger is stirred for 6 hours at 50 °C, reducing the palladium level to ≤ 2 µg/g as measured by inductively coupled plasma mass spectrometry after microwave digestion. The treated solution is concentrated under vacuum at ≤ 35 °C to a final residual volume of 2.5 volumes relative to the substrate mass, and the product is crystallized by the addition of n-heptane (8 volumes) over 4 hours with a linear cooling ramp from 45 °C to 0 °C. The crystalline tripeptide intermediate, isolated by centrifugation and dried at 40 °C under 50 mbar for 24 hours, exhibited an assay of 98.8% (qNMR, maleic acid internal standard) and an enantiomeric excess of > 99.9% determined by chiral supercritical fluid chromatography on a Chiralpak AD-H column (250 × 4.6 mm, 5 µm) with a mobile phase of CO2–methanol (80:20 v/v) containing 0.1% isopropylamine. This intermediate serves as the penultimate fragment for a second-generation hepatitis C virus NS3/4A macrocyclic protease inhibitor that entered Phase III trials.Anhydride-Mediated Activation for a Fragment-Based Covalent Inhibitor LibraryCombinatorial chemistry campaigns targeting a cysteine protease with a conserved active-site thiol have exploited the steric and electronic bias of the (4S)-methyl substituent on the pyrrolidine ring to direct inhibitor conformation. In a representative high-throughput parallel synthesis, 96-well deep-well plates were charged with 0.15 mmol of the title compound in each well, and the carboxylic acid was pre-activated with 1.25 equivalents of isobutyl chloroformate and 1.5 equivalents of N-methylmorpholine in anhydrous dichloromethane at −15 °C under a nitrogen blanket. After 30 minutes of activation, a solution containing 0.10 mmol of an amine-bearing warhead fragment (e.g., a vinyl sulfonamide or acrylamide scaffold) was dispensed, and the reaction was agitated at 22 °C for 16 hours. The Boc group was subsequently removed without intermediate purification by adding 0.5 mL of a pre-cooled 4 M HCl in dioxane solution, and the mixture was agitated for 2 hours at 20 °C followed by evaporation in a Genevac centrifugal evaporator. The crude tertiary amide products were purified by mass-directed preparative LC-MS using a Waters XBridge C18 column (19 × 100 mm, 5 µm) and a gradient of acetonitrile in 10 mM ammonium bicarbonate buffer (pH 8.0), yielding library members with an average purity of ≥ 95% by HPLC-UV at 254 nm. A challenge with this synthetic sequence is the formation of 3–5% of the 2-epi diastereomer during activation, traced to α-proton abstraction by excess N-methylmorpholine; this was mitigated by reducing the base to 1.05 equivalents and lowering the activation temperature to −25 °C, which kept epimerization below 0.5%. The structure–activity relationship data derived from these covalently modified proteins were cross-validated with X-ray crystallography, confirming that the (4S)-methyl group occupies a hydrophobic sub-pocket in the S2 binding region, a feature that can only be incorporated using the enantiomerically pure title compound as the input chiral building block.
How Does Metal Scavenger Selection Impact Copper Contamination in Click Chemistry Conjugates?A manufacturing route to a triazole-linked glycopeptide antibiotic conjugate merged the title compound-derived alkyne fragment with a carbohydrate azide via copper-catalysed azide-alkyne cycloaddition (CuAAC). The (4S)-methylpyrrolidine scaffold was functionalised at the nitrogen after Boc deprotection with 4-pentynoic acid using HATU coupling to introduce the terminal alkyne handle. The CuAAC step was performed in a tertiary solvent system of tert-butanol–water–dichloromethane (2:1:1 v/v/v) containing 0.15 equivalents of copper(II) sulfate pentahydrate and 0.30 equivalents of sodium ascorbate at 35 °C. Following 18-hour reaction time, the resulting solution contained 800–1200 ppm dissolved copper, exceeding the ICH Q3D permitted daily exposure for oral administration routes. Three downstream scavenging workflows were benchmarked on a 50-g pilot scale: Method A utilised 5% w/w QuadraSil MP resin (macroporous polystyrene-bound ethylenediaminetetraacetic acid) stirred for 12 hours; Method B employed a 10% w/v aqueous ammonia wash (pH 9.5) repeated three times; and Method C passed the concentrate through a Zeba size-exclusion desalting column pre-equilibrated with 0.5 M ethylenediaminetetraacetic acid disodium salt. Copper content was quantified by graphite furnace atomic absorption spectroscopy with a limit of quantitation of 0.5 µg/g. Only Method A achieved residual copper below the 100 µg/g threshold mandated for the subsequent lyophilisation and terminal sterilisation, yielding 12 µg/g copper in the final conjugate with 99.4% mass balance. The campaign highlighted that without diligent metal clearance, the copper adducts had catalysed oxidative degradation of the triazole ring during accelerated stability storage at 40 °C/75% RH, leading to a 3.5% total impurity increase over 6 months. The scavenger-treated lot maintained individual unspecified impurities at ≤ 0.10% over the same period, satisfying ICH Q1A(R2) stability criteria.
Auxiliary-Driven Diastereomeric Salt Resolution to Recover Specification-Grade Material from a Mother Liquor StreamBatch-to-batch variability in the diastereomeric purity of commercially sourced (2S,4S)-1-(tert-butoxycarbonyl)-4-methylpyrrolidine-2-carboxylic acid occasionally delivers lots with an enantiomeric excess of 97.0–98.5%, below the ≥ 99.0% threshold required for a parenteral-grade active pharmaceutical ingredient submission. An industrial chiroptical upgrade was implemented by exploiting classical resolution with (1R,2R)-(−)-pseudoephedrine as the resolving agent. The lower-ee substrate (20.0 kg, 97.2% ee) was dissolved in 160 litres of ethyl acetate–acetone (3:1 v/v) at 60 °C, and 0.55 equivalents of the pseudoephedrine base were added in one portion. The clear solution was seeded with 0.5% w/w of previously isolated diastereomerically pure salt and cooled according to a cubic cooling profile (0.15 °C/min ramp to 20 °C, followed by a hold of 2 hours, then 0.05 °C/min ramp to 5 °C). The precipitated salt was filtered, washed with cold ethyl acetate, and suspended in 1 M hydrochloric acid to liberate the free acid, which was back-extracted with methylene chloride. After drying over anhydrous sodium sulfate and concentration, 15.8 kg of title compound was recovered with a chemical yield of 79% and an enantiomeric excess of 99.8% as determined by chiral gas chromatography on a CP-Chirasil-Dex CB column (25 m × 0.25 mm, 0.25 µm) using a temperature ramp from 120 °C to 200 °C at 3 °C/min. The mother liquor enriched in the undesired (2R,4R)-enantiomer was basified and the resolving agent recovered by distillation for reuse in subsequent cycles. In-process controls for residual (1R,2R)-(−)-pseudoephedrine in the final product employed an ion-pairing reversed-phase HPLC method with a limit of detection of 10 ppm, ensuring that the residual amine level remained below the 50 ppm limit established through a toxicological qualification study in accordance with ICH M7 for a DNA-reactive (mutagenic) impurity classification.What Limits the Throughput of Continuous-Flow Boc Deprotection for Process Intensification?Transitioning from batch-mode Boc removal with 4 M HCl in dioxane to a continuous-flow protocol was evaluated to reduce cycle time and manual handling of corrosive reagents for an advanced HIV protease inhibitor intermediate. The protected pyrrolidine acid (1.0 M in methanol) was combined with a 2.5-fold molar excess of acetyl chloride in methanol generated in-line to produce anhydrous HCl, and the mixture was pumped through a 10 mL perfluoroalkoxy alkane coiled tube reactor (internal diameter 2.0 mm) immersed in a thermostatic oil bath at 50 °C. Back-pressure regulation set to 6 barg suppressed bubble nucleation and ensured a homogeneous single-phase regime. Residence time distribution studies using a step-input tracer (acetone) indicated a plug-flow behaviour with an axial dispersion coefficient below 0.02, allowing the synthesis of the free amine hydrochloride with 99.7% conversion at a steady-state flow rate of 4.5 mL/min. The limiting factor to higher throughput was the precipitation of the hydrochloride salt within the reactor coil at product concentrations exceeding 0.6 M; salt accretion on the inner wall eventually caused a pressure excursion of > 15 barg and triggered the safety interlock. Mitigation involved addition of 15% v/v acetonitrile as a co-solvent to increase salt solubility and incorporation of an inline 100 µm wedge-wire filter to capture particulate matter downstream of the reactor. The continuous-deprotected amino acid solution was directly telescoped into the next amide coupling step, eliminating an isolation and drying operation that previously required 48 hours and reducing residual Boc-protected starting material carryover to ≤ 0.15% by HPLC. This process intensification outcome, operated under ICH Q13 continuous manufacturing principles, decreased the overall process mass intensity from 42 to 28 kg/kg active pharmaceutical ingredient. |
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The (2S,4S)-1-(tert-butoxycarbonyl)-4-methylpyrrolidine-2-carboxylic acid is supplied as a white to off-white crystalline solid with a molecular formula C₁₁H₁₉NO₄, a molecular weight of 229.27 g mol⁻¹, a melting range of 122–125 °C, and a specific rotation [α]D20 = −58° ± 2° (c = 1, CH₃OH). The material is routinely released at a purity of ≥98.5% by reversed-phase HPLC (210 nm) and an enantiomeric excess of ≥99.5% by chiral stationary-phase HPLC (Chiralpak® IA column, n-hexane/isopropanol/trifluoroacetic acid 90/10/0.1). The amine protecting group, tert-butoxycarbonyl (Boc), is installed on the secondary nitrogen of the pyrrolidine ring, while the (2S,4S) absolute configuration enforces a cis relationship between the carboxy function and the 4-methyl substituent. This stereochemistry distinguishes the product from the more widely exploited (2S,4R) epimer, in which the 4-methyl group occupies a trans disposition relative to the carboxylate, and from racemic or enantiopure Boc-4-methylproline preparations where the methyl-bearing carbon is not stereodefined.
| Parameter | (2S,4S)-Boc-4-Me-Pro-OH | (2S,4R)-Boc-4-Me-Pro-OH | Boc-Pro-OH | Boc-4-Me-Pro-OH (racemic) |
|---|---|---|---|---|
| Purity (HPLC, 210 nm) | ≥98.5% | ≥98.0% | ≥99.0% | ≥97.0% |
| Enantiomeric excess | ≥99.5% | ≥99.0% | — | — |
| Melting range (°C) | 122–125 | 128–131 | 132–136 | 118–124 |
| [α]D20 (c = 1, MeOH) | −58° ± 2° | −70° ± 2° | −60° ± 2° (lit.) | 0° ± 5° |
| Epimerization in model coupling (%) | 0.15 | 0.30 | 1.10 | — |
| Trans-amide rotamer population (%) | 85 | 55 | 65 | — |
| Test | Method | Specification | Result |
|---|---|---|---|
| Assay (anhydrous basis) | HPLC, area % | 98.0–102.0% | 99.2% |
| Chiral purity | HPLC, Chiralpak IA | NMT 0.5% (2S,4R) epimer | 0.08% |
| Residual DMF | GC headspace | ≤ 880 ppm | 120 ppm |
| Chloride (as counterion) | Ion chromatography | ≤ 0.05% | 0.01% |
| Heavy metals (total) | USP <231> Method II | ≤ 20 ppm | < 5 ppm |
