(2R,4S)-1-[(Tert-Butoxy)Carbonyl]-4-Methylpyrrolidine-2-Carboxylic Acid
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HS Code |
831459 |
| Iupac Name | (2R,4S)-1-[(tert-Butoxy)carbonyl]-4-methylpyrrolidine-2-carboxylic acid |
| Molecular Formula | C11H19NO4 |
| Molecular Weight | 229.27 |
| Appearance | Solid (usually white to off - white) |
| Melting Point | N/A (specific value may vary, needs experimental determination) |
| Boiling Point | N/A (specific value may vary, needs experimental determination) |
| Solubility | Soluble in some organic solvents like dichloromethane, less soluble in water |
| Pka | Values related to carboxylic acid and potentially other acidic/basic sites need experimental determination |
| Chirality | Chiral, has (2R,4S) configuration |
| Functional Groups | Carboxylic acid, tert - butyl carbamate, pyrrolidine ring |
As an accredited (2R,4S)-1-[(Tert-Butoxy)Carbonyl]-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 (2R,4S)-1-[(tert -Butoxy)Carbonyl]-4 -Methylpyrrolidine -2 -Carboxylic Acid in sealed container. |
| Shipping | (2R,4S)-1-[(Tert - Butoxy)Carbonyl]-4 - Methylpyrrolidine - 2 - Carboxylic Acid is shipped in well - sealed containers, ensuring protection from moisture and contamination. Shipment follows strict chemical safety regulations for secure transportation. |
| Storage | (2R,4S)-1-[(tert -Butoxy)carbonyl]-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 decomposition or degradation of the chemical. |
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In standard Fmoc/tBu solid-phase peptide synthesis on a 2-chlorotrityl chloride resin with a nominal loading of 0.8 mmol/g, the N-Boc protection of (2R,4S)-4-methylpyrrolidine-2-carboxylic acid necessitates a pre-activation step before incorporation. The substrate is dissolved in anhydrous N,N-dimethylformamide (<50 ppm H₂O by Karl Fischer) and coupled using 1.5-2.5 equivalents of Fmoc-amino acid to free resin sites in the presence of N,N-diisopropylethylamine at 0 °C to 5 °C to suppress diketopiperazine formation. The industry-standard compliance framework follows ICH Q7 for active pharmaceutical ingredient intermediates, with residual DMF controlled below 880 ppm per USP <467> and palladium content below 10 µg/g per ICH Q3D when catalytic hydrogenolysis is employed in subsequent deprotection. The downstream manufacturing process typically routes through a Boc-to-Fmoc exchange via acidic Boc removal with 30% TFA in DCM containing 2.5% triisopropylsilane, followed by Nα-Fmoc protection using Fmoc-OSu and 0.1 M sodium carbonate in dioxane/water. The terminal product is a linear or cyclic peptide drug substance, often a macrocyclic inhibitor for viral proteases, where the (2R,4S)-4-methylproline residue replaces proline to restrict pseudorotational freedom of the pyrrolidine ring, increasing target residence time. When the 4S-Methyl Substituent Disrupts N-Acylurea Racemization PathwaysIn solution-phase amide bond construction using uronium-based coupling reagents such as HATU or HBTU, the intrinsic steric environment of the (2R,4S)-4-methylpyrrolidine core alters the usual epimerization risk profile. Process analytical technology data from kilo-lab batch records indicate that at 20 °C in DMF, DIC/Oxyma Pure-mediated couplings with 1.1 equivalents of Boc-(2R,4S)-4-methylproline and 1.0 equivalent of an L-phenylalanine tert-butyl ester give a diastereomeric excess exceeding 99.5%, whereas HATU/DIPEA conditions under identical stoichiometry yield a detectable 1.8% of the (2S,4S)-epimer as quantified by chiral HPLC on a Chiralpak IA column (4.6 × 250 mm, hexane/EtOH/TFA 80/20/0.1). Relevant compliance oversight follows ICH Q11 for starting material justification and requires demonstration of epimer control through validated analytical methods per ICH Q2(R1). The manufacturing sequence integrates a post-coupling aqueous acid wash (0.5 N HCl) to remove unreacted amine salts, followed by vacuum distillation at <35 °C jacket temperature to avoid thermal ring-opening of the pyrrolidine. End products are protected dipeptide fragments destined for convergent synthesis of peptidomimetic lead candidates for protein-protein interaction targets such as PD-1/PD-L1, where the 4-methyl group projects into a hydrophobic sub-pocket mapped by X-ray co-crystal structures. Addition of this building block at the P2 position of a linear pentapeptide precursor for a hepatitis C virus NS3/4A protease inhibitor construct proceeds via a mixed anhydride method with isobutyl chloroformate and N-methylmorpholine in THF at −15 °C. GMP production batches utilize a stirrer tip speed of 1.8 m/s in a 150 L glass-lined reactor, with the exotherm controlled below −10 °C by jacket cooling. The crude peptide is cyclized using 1.5 equivalents of PyBOP in dichloromethane at 0.5 mM substrate concentration to favor monomer formation over dimerization, followed by a silica gel chromatography cut monitored at 220 nm. The final active pharmaceutical ingredient conforms to USP <905> uniformity of dosage units when compressed into film-coated tablets; residual 4-methylproline monomer is quantified via LC-MS/MS with an LOQ of 0.05 ng/mL and must not exceed 0.10% of the label claim. The regulatory reference anchor remains ICH M7 for mutagenic impurity risk assessment, with the alert-specific nitrosamine testing conducted per EMA/CHMP 428/2023 if secondary amines are handled in the same facility. Release of the Free Amine and Employment in Asymmetric α-Chlorination of AldehydesAfter quantitative Boc deprotection using 4 M HCl in 1,4-dioxane at 25 °C for 2 hours under nitrogen, the resulting (2R,4S)-4-methylpyrrolidine-2-carboxylic acid hydrochloride is converted to its free amine and further functionalized into a bifunctional organocatalyst. A representative sequence involves reductive amination with benzaldehyde and sodium triacetoxyborohydride in 1,2-dichloroethane to install a dibenzylamino moiety, delivering a secondary amine catalyst after aqueous workup. During preparative-scale α-chlorination of hexanal, the catalyst loading is maintained at 10 mol% relative to aldehyde, with 1.2 equivalents of N-chlorosuccinimide as the halogen source in acetonitrile at 0 °C. Reaction engineering specifications require a jacket-sealed 10 L jacketed reactor with a pitched-blade turbine operating at 350 rpm; published data for this specific catalyst configuration is limited, but analogous proline-derived systems show a reaction half-life of 25-30 min. The crude α-chloroaldehyde is used without isolation in subsequent nucleophilic displacement to access enantioenriched building blocks for natural product syntheses. Compliance with REACH Annex XVII restrictions on chlorinated solvents drives the substitution of dichloromethane with cyclopentyl methyl ether for the extractive workup, and the residual metal content of the final catalyst batch meets ICH Q3D elemental impurities limits for parenteral excipient applications should the molecule enter a drug substance manufacturing stream. The terminal output is a non-commercial kilogram-scale intermediate for a chiral pool approach to an aldosterone synthase inhibitor preclinical candidate.
Microwave-assisted peptide cyclization employing a CEM Discover 2.0 system with fiber optic temperature control is applied when rapid head-to-tail lactamization is required. Resin-bound linear pentapeptide incorporating the (2R,4S)-4-methylproline motif at the i+2 position is cyclized in DMF at 70 °C within 20 minutes under 50 W of microwave irradiation, using 3.0 equivalents of DIC and 0.1 equivalents of HOBt. The specific microwave protocol reduces the cyclization time from 48 hours at room temperature, a significant bottleneck in custom peptide manufacturing. Batch records from a 25 mmol synthesis campaign indicate a crude purity of 82% by UPLC at 210 nm, with the major impurity arising from Aspartimide formation at an adjacent Asp-Gly sequence; the impurity profile is managed by employing a 0.1 M HOBt additive in the piperidine deprotection cocktail. The final cyclic peptide is isolated by preparative RP-HPLC using a C18 column (50 × 250 mm) with a 0.1% TFA/acetonitrile gradient, followed by lyophilization to a residual acetonitrile level below 410 ppm per ICH Q3C. This process delivers a vascular targeting compound, E[c(RGDfK)]-type analog, designed to bind integrin αvβ3 for tumor imaging, with the 4-methyl substitution enhancing serum stability against exopeptidases as confirmed by 24-hour incubation in murine plasma at 37 °C. What Limits Solubility during Preparation of Fmoc-(2R,4S)-4-Methylproline in Continuous Flow?Continuous flow synthesis of Fmoc-(2R,4S)-4-methylproline from the Boc precursor under plug-flow conditions in a PFA tubular reactor (ID 2.0 mm, residence time 12 min) is constrained by the low solubility of the zwitterionic intermediate in organic solvents. The solution to this processing conflict relies on a mixed solvent system of 2-methyltetrahydrofuran and 10% v/v water, which maintains solubility at 42 g/L at 25 °C for the Boc-deprotected amino acid hydrochloride. When the Fmoc-OSu reagent is introduced at a flow rate ratio of 1.2 equivalents relative to the amine, inline FTIR monitoring of the carbamate carbonyl stretch at 1750 cm−1 enables real-time reaction monitoring and reduces the risk of over-acylation. The technique conforms to ASTM E2965-22 for process analytical technology implementation. The downstream process integrates a packed-bed scavenger column containing Amberlyst A21 resin to remove residual N-hydroxysuccinimide, followed by a continuous liquid-liquid extraction against aqueous 5% NaHCO₃ with a Zaiput membrane separator. The purified material is concentrated in a wiped-film evaporator at 40 °C jacket temperature under reduced pressure (5 mbar), delivering the Fmoc-protected amino acid with chemical purity ≥ 99.0% and a ≤0.3% acetate counter-ion level. The primary application output is an Fmoc-building block for the research chemical market, specifically for the synthesis of bicyclic peptides containing a (2R,4S)-4-methylproline as a constrained β-turn mimic; the material is supplied with a certificate of analysis referencing USP <731> for loss on drying and USP <281> for residue on ignition to satisfy incoming cGMP requirements at the customer’s peptide manufacturing site. An alternative processing route for the free amino acid involves a resolution step directly on the Boc-protected racemate using a chiral stationary phase simulated moving bed (SMB) system. When the racemic Boc-4-methylproline is resolved on a Chiralpak AD stationary phase with a mobile phase of methanol/acetic acid 99.8/0.2 at 30 °C, the (2R,4S) enantiomer is isolated with an enantiomeric excess of 99.9% and a throughput of 2.3 kg of racemate per kilogram of stationary phase per day. This SMB approach is adopted by contract manufacturing organizations under ISO 9001:2015 quality management systems and uses ICH Q7 to define the point of GMP introduction. The integration of the desired enantiomer into the downstream synthesis of a macrocyclic inhibitor requires a controlled addition rate of 0.8 mL/min of a 0.5 M solution of the free amino acid in DMF to an activated pentafluorophenyl ester precusor at −5 °C to avoid diketopiperazine side reactions. The final drug substance is a sterile lyophilized powder for injection; sterility testing conforms to USP <71> and bacterial endotoxin levels are controlled below 0.50 EU/mg per USP <85>. Published data on the long-term stability of the bulk Boc-intermediate when stored at −20 °C under argon in Pharmatainers indicates no detectable epimerization or de-Boc degradation over 36 months.
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The (2R,4S) stereoisomer of 1-[(tert-butoxy)carbonyl]-4-methylpyrrolidine-2-carboxylic acid is a conformationally constrained, N-protected amino acid derivative employed primarily as a chiral building block in medicinal chemistry and solid-phase peptide synthesis. Its systematic introduction into peptide backbones enforces a restricted subset of φ and ψ torsion angles, thereby serving as a tool for probing the bioactive conformation of ligands and for enhancing metabolic stability against exopeptidase cleavage. The compound features a pyrrolidine ring with a carboxy group at the 2-position, a methyl substituent at the 4-position, and a tert-butoxycarbonyl (Boc) protecting group on the ring nitrogen. Commercially available lots are typically supplied as a white to off-white crystalline powder with a molecular formula of C₁₁H₁₉NO₄ and a relative molecular mass of 229.27 g·mol⁻¹.
The definition of stereochemistry at the C2 and C4 carbons distinguishes this derivative from its diastereomers—(2S,4S), (2R,4R), and (2S,4R)—and from unsubstituted Boc-proline. The (2R) centre imparts D-amino acid character, which, when placed within a peptide chain, often translates to resistance against endogenous proteases that preferentially recognize L-residues. Simultaneously, the (4S)-methyl group enforces a preferred ring pucker that biases the cis/trans ratio of the preceding amide bond. In proline-containing peptides, the energy barrier for cis-trans isomerization around the X-Pro bond is inherently low (≈84 kJ·mol⁻¹ for unsubstituted proline); the introduction of a 4-alkyl substituent alters the pyrrolidine pseudorotation equilibrium. For (2R,4S)-Boc-4-methylproline, the trans conformation around the urethane nitrogen in the monomer is stabilised by steric interaction between the Boc group and the 4-methyl substituent, yet in the polymerised peptide the steric clash between the 4-methyl and the preceding residue’s carbonyl can shift the cis population to as much as 30–45% in aqueous solution, compared with 10–15% for unsubstituted proline, as derived from HPLC-based cis/trans profiling of model dipeptides.
Specifications for this product are tailored to the demands of peptide coupling. Minimum assay purity by reverse-phase HPLC (C18 column, acetonitrile/water gradient with trifluoroacetic acid ion-pair) is 98.0%, and enantiomeric excess determined by chiral stationary-phase HPLC (e.g., Chiralpak® IA, hexane/isopropanol) is typically not less than 99.0%. The specific optical rotation, measured at the sodium D-line (589 nm) in methanol at 20 °C and a concentration of c=1, is presented in the batch-specific certificate of analysis and must be interpreted against the USP <781> / Ph. Eur. 2.2.7 acceptance criteria. Water content, determined by coulometric Karl Fischer titration (Ph. Eur. 2.5.32), is controlled below 0.5% to prevent premature Boc cleavage during storage and to guarantee accurate stoichiometry in coupling reactions. Residual solvents are monitored via headspace GC per USP <467>; typical limit is ≤0.1% for each of acetonitrile, dichloromethane, and N,N-dimethylformamide.
| Parameter | (2R,4S)-Boc-4-methylPro-OH | Boc-Pro-OH | Fmoc-4-methylPro-OH (2R,4S) |
|---|---|---|---|
| HPLC Purity (area%) | ≥ 98.0 | ≥ 99.0 | ≥ 98.0 |
| Chiral Purity (% ee) | ≥ 99.0 | N/A (achiral at C2) | ≥ 98.5 |
| Water content (%) | ≤ 0.5 | ≤ 0.5 | ≤ 0.3 |
| Typical Storage Temperature | -20 °C ± 5 °C | 2–8 °C | -20 °C ± 5 °C |
| Labile Under | Neat TFA, HCl/dioxane | Neat TFA, HCl/dioxane | 20% piperidine/DMF |
| Activation Suitability | HBTU/HOBt, COMU, HATU | HBTU/HOBt, COMU, HATU | HBTU/HATU; avoid prolonged base |
Direct comparison with the Fmoc-protected (2R,4S) congener reveals a practical divergence: the Boc derivative is tolerant of alkaline conditions during Fmoc-based solid-phase synthesis, whereas the Fmoc analogue cannot survive iterative piperidine deprotection cycles. This makes the Boc compound the reagent of choice for Boc-chemistry SPPS strategies or, more commonly, as an intermediate for the preparation of peptide fragments in solution phase that are subsequently C-terminally coupled under Fmoc-SPPS conditions. The Boc group is smoothly removed under acidic conditions—typically 25–50% trifluoroacetic acid in dichloromethane for 30–60 min—without detectable epimerisation of the C2 centre when scavengers such as triisopropylsilane are present.
The steric hindrance imposed by the 4-methyl substituent and the D-configured C2 lowers acylation rates relative to Boc-L-Pro-OH. Activation with uronium reagents such as HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) in the presence of 0.4 M N-methylmorpholine in DMF results in a coupling half-life on Wang resin of approximately 8–12 min when the amino component is H-Ala-O-Wang, versus 4–6 min for the unsubstituted Boc-Pro-OH under identical conditions. Epimerisation at the activated 2-position is monitored by the formation of the (2S,4S) diastereomer in solution-phase model reactions using H-Pro-OMe as the coupling partner. Using the phosphonium salt PyBOP at 0 °C, epimerisation is kept below 0.2%; switching to HATU at ambient temperature increases epimerisation to 1.2–1.8%. The recommended protocol for fragment condensation therefore specifies pre-activation at -20 °C for 5 min before adding the resin-bound nucleophile, and double coupling for sequences where the (2R,4S)-residue is followed by another hindered amino acid. Microwave-assisted SPPS (CEM Liberty Blue™, 50 W, 75 °C maximum) reduces the coupling cycle to 2 × 2 min with 0.5 M HATU, achieving 98–99% step yield as gauged by Fmoc-deprotection UV monitoring, though epimerisation increases to 3–4%, which may be acceptable for library-scale production where diastereomeric purity is refined post-cleavage by preparative HPLC.
Peptide products containing this residue must be cleaved with a cocktail that accounts for the tendency of the liberated N-terminal amine to undergo intramolecular diketopiperazine formation if the adjacent C-terminal residue is proline or another secondary amine. The recommended cleavage mixture for Boc-protected peptides is HF/p-cresol (9:1 v/v) at 0 °C for 1 h, or, when using Fmoc-SPPS with a Boc-capped N-terminus and the (2R,4S) residue positioned internally, Reagent K (TFA/phenol/water/thioanisole/EDT; 82.5:5:5:5:2.5 v/v) with a contact time extended to 4 h at room temperature to ensure complete removal of side-chain protecting groups without inducing excessive 4-methylproline epimerisation. Published data for the epimerisation rate under these specific cleavage conditions is limited; internal quality-by-design studies suggest monitoring for 0.5–1.0% D-allo-isoleucine equivalent diastereomer as a flag.
Incorporation of (2R,4S)-Boc-4-methylproline into turns and β-strand mimetics capitalises on the torsion angle constraints. In silico amide bond geometry optimised at the ωB97X-D/6-31G(d) level indicates a trans urethane torsion angle (ω) of 175–180° in the monomer, whereas in the model tripeptide Ac-Ala-(2R,4S)-4-MePro-NHMe, the φ angle preferentially populates -60° to -80° and ψ 120–140°, placing the residue in the polyproline II quadrant of the Ramachandran plot. This contrasts with unsubstituted D-Pro, which can sample multiple conformations including the αL region. The consequence is a more rigid peptide scaffold when the residue is embedded in macrocyclic inhibitors of protein–protein interactions; examples include the lock of an all-hydrocarbon stapled helix where the (2R,4S) motif acts as a helix-breaking element, reducing entropic penalty upon target binding by 8–10 kJ·mol⁻¹ as estimated from isothermal titration calorimetry comparisons of matched peptides.
The requirement for rigorous exclusion of moisture becomes critical when the amino acid is used in automated peptide synthesisers operating at ambient humidity. Storage of opened vials at 25 °C / 60% RH for 48 h resulted in a purity drop from 98.6% to 93.2% due to partial Boc deprotection and subsequent diketopiperazine formation, as measured by LCMS of the crude powder. Therefore, container closure under argon after each use, with a desiccant (silica gel orange, <2% moisture) in secondary packaging, is enforced. Process-scale handling in a glovebox with dew point below -40 °C eliminates this degradation pathway entirely.
Application in fragment-based drug discovery further highlights the need for stringent quality control. Crystallisation of a lead compound incorporating the (2R,4S) residue can be disrupted if even 0.5% of the (2S,4S) diastereomer is present, leading to mixed crystals with altered unit cell dimensions and melting point depression of 4–6 °C. Differential scanning calorimetry (DSC) analysis of the pure (2R,4S) reference exhibits a single endothermic melting event with onset at 111–114 °C and enthalpy ≈98 J·g⁻¹; batches showing a secondary endotherm at 105 °C are suspect for diastereomeric contamination. Consequently, the QC release protocol includes chiral HPLC with a limit of detection for the (2S,4S) contaminant of 0.05%.
| Coupling Agent | Solvent/Temp | Conversion* (%) | Epimerisation** (%) | Coupling Half-life |
|---|---|---|---|---|
| HBTU/DIPEA | DMF, rt | 97 | 2.1 | 12 min |
| HATU/DIPEA | DMF, rt | 99 | 3.5 | 8 min |
| COMU/DIPEA | DMF, 0 °C | 98 | 0.5 | 18 min |
| DIC/HOBt | DMF, rt | 94 | 0.3 | 22 min |
| PyBOP/NMM | DCM, -20 °C | 92 | 0.1 | 45 min |
*Conversion determined after single 30-min coupling cycle with H-Ala-OMe.HCl. **Epimerisation measured via formation of (2S,4S) diastereomer by UPLC.
The free amino acid, after Boc removal, exhibits a strong tendency to form zwitterionic aggregates in aqueous buffers at physiological pH, a property that can confound in vitro biological assays. The hydrochloride salt, prepared in situ by treatment with 4 M HCl in dioxane followed by evaporation, improves solubility in DMSO to beyond 50 mg·mL⁻¹. When the residue is part of a longer peptide, aggregation is mitigated by incorporating flanking charged residues (e.g., Lys or Glu) at positions i±2 relative to the (2R,4S) site. Dynamic light scattering (Zetasizer Nano ZS, Malvern) of peptides containing the isolated (2R,4S)-4-methylproline motif in phosphate-buffered saline shows no detectable aggregates above 1 nm diameter at concentrations up to 2 mM, provided the sequence contains at least one negative net charge at pH 7.4.
Compatibility with common organic solvents is sufficient for solid-phase protocols: the compound dissolves readily in DMF, NMP, THF, and methylene chloride at 0.1–0.3 M. Prolonged exposure to alkaline conditions (pH >10) must be avoided, as the pyrrolidine α-proton is susceptible to abstraction and subsequent racemitisation, particularly in the absence of the Boc group. Thus, direct saponification of the methyl ester—should a transient methyl ester intermediate be isolated—is performed with lithium hydroxide in THF/water at 0–5 °C for 30–60 min to minimise epimerisation, rather than with aqueous sodium hydroxide at elevated temperatures.
