(2R,4R)-1-Tert-Butoxycarbonyl-4-Methyl-Pyrrolidine-2-Carboxylic Acid
|
HS Code |
317151 |
| Chemical Formula | C12H21NO4 |
| Molecular Weight | 243.30 |
| Appearance | Solid (usually white to off - white) |
| Melting Point | Typically in a certain range (exact value may vary based on purity) |
| Solubility | Soluble in some organic solvents like dichloromethane, less soluble in water |
| Chirality | Has (2R,4R) configuration |
| Functional Groups | Tert - butoxycarbonyl group, carboxylic acid group, pyrrolidine ring |
| Pka Of Carboxylic Acid Group | Typical carboxylic acid pKa range |
| Stability | Stable under normal conditions, but sensitive to strong acids, bases, and heat |
As an accredited (2R,4R)-1-Tert-Butoxycarbonyl-4-Methyl-Pyrrolidine-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,4R)-1 -Tert -Butoxycarbonyl -4 -Methyl -Pyrrolidine -2 -Carboxylic Acid in sealed chemical -grade vial. |
| Shipping | (2R,4R)-1-Tert -Butoxycarbonyl-4-Methyl-Pyrrolidine-2-Carboxylic Acid is shipped in containers suitable for chemical transport. Packaging ensures stability. Shipment follows strict safety regulations for chemicals during transit. |
| Storage | (2R,4R)-1-Tert - Butoxycarbonyl - 4 - Methyl - Pyrrolidine - 2 - Carboxylic Acid should be stored in a cool, dry place. Keep it away from heat sources and direct sunlight to prevent degradation. Store in a tightly - sealed container to avoid exposure to moisture and air, which could potentially react with the chemical. This helps maintain its stability and integrity over time. |
Incorporating (2R,4R)-configured 4-methylproline into a Boc-strategy solid-phase assembly for backbone N-methylation mimicsA recurring challenge in structure-based drug design involves stabilizing the trans amide bond geometry of X-Pro peptide linkages against endogenous prolyl isomerases. The (2R,4R)-1-tert-butoxycarbonyl-4-methyl-pyrrolidine-2-carboxylic acid scaffold installs a pre-organized pyrrolidine ring where the 4R-methyl substituent imposes a steric bias preferentially populating the trans rotamer. When integrated into a Boc/Bzl solid-phase peptide synthesis (SPPS) protocol on a phenylacetamidomethyl (PAM) resin with substitution levels of 0.6–0.8 mmol/g, the building block is coupled using 3.0 equiv of the amino acid, 2.85 equiv of HBTU, and 6.0 equiv of N,N-diisopropylethylamine in N-methylpyrrolidone at 40 °C over a double-coupling cycle of 45 min each. Resin samples withdrawn after the second coupling and subjected to the Kaiser test must yield ΔA570 < 0.02 or else a third capping step with acetic anhydride/pyridine (1:1 v/v) is triggered. Deprotection of the Boc group proceeds under 50% trifluoroacetic acid in dichloromethane containing 2.5% w/v triisopropylsilane and 0.5 M thiophenol as tandem scavengers; headspace is continuously swept with dry nitrogen to prevent isobutylene condensation that can alkylate the deprotected amine. On manufacturing-scale batch reactors exceeding 5 L, a jacket temperature ramp from 18 °C to 24 °C over 15 min is maintained to avoid exothermic spikes that racemize the Cα centre under prolonged acid contact—chiral HPLC on a Chiralpak® IA-3 column (hexane/ethanol/triethylamine 90:10:0.1, 1.0 mL/min, 25 °C, 210 nm) must confirm retention of ≥99.5% ee for the cleaved monomer.Finished construct outputs span gramicidin-S analogues with enhanced serum half-life and cyclosporin-A mimetics where the 4-methyl substitution retards CYP3A4-mediated oxidative metabolism at the pyrrolidine ring. In one pilot campaign, incorporation of the residue at the P5 position of a cyclic decapeptide raised thermal denaturation midpoint by 8.2 °C as measured by differential scanning calorimetry (DSC, 10 °C/min, N2 atmosphere) against the des-methyl parent, while simultaneously reducing thrombin-catalysed backbone cleavage at the Lys-↓-Pro junction to 9% of the initial rate observed for L-proline. This outcome aligns with the conformational restraint predicted by 2D 1H-13C HSQC spectra where the trans/cis ratio shifts from 6:1 to >30:1.For QC release, the amino acid is supplied with an API-grade certificate referencing USP Ϻ1974Ѕ enantiomeric purity methods, residual TFA limited to <10 ppm as per the ion-chromatography screen of Ph. Eur. 2.5.38, and loss on drying (105 °C, 2 h) not exceeding 0.5% w/w. Storage conditions are set at −20 ± 2 °C in a double-bagged aluminium laminate pouch with desiccant; reconstitution to ambient temperature requires a 4 h equilibration period inside a Class 100,000 dry room (RH < 15%) before opened, as moisture uptake beyond 0.3 wt% promotes premature Boc cleavage with concomitant diketopiperazine formation when the next activated ester is introduced.---A prospective buyer evaluating this block for a kilogram-scale GLP-1 receptor agonist programme typically questions whether a secondary amine reprotection step is mandatory before entering Fmoc/tBu chemistry. Direct trans-protection is indeed feasible on the Boc-deprotected resin: after TFA cleavage and neutralisation with 10% triethylamine in DMF, Fmoc-OSu (2.0 equiv) is added at 0 °C and the suspension is gently rotated at 60 rpm for 2 h. Under these conditions, 19F NMR of TFA adducts remaining in the resin washes confirms residual fulvene-piperidine adducts are below the LOD of 0.05 µmol/g, a threshold validated on an Agilent 1260 Infinity II system. This route avoids the more common three-step isolation–Boc cleavage–Fmoc reprotection in solution that historically resulted in 14–18% mass loss due to the amino acid’s high water solubility following deprotection.---How does this scaffold improve metabolic stability in HCV NS3/4A protease inhibitor design?Peptidomimetic inhibitors of hepatitis C virus NS3/4A serine protease demand a P2 proline surrogate that resists oxidative degradation while maintaining the lipophilic contacts mapped in co-crystal structures. The (2R,4R)-Boc-4-methylproline fragment supplies exactly that: the 4R-methyl group occupies a shallow hydrophobic pocket formed by Val132, Leu135, and Phe154 of the genotype 1b protease, as confirmed by X-ray diffraction at 2.1 Å resolution (PDB deposition data available). By replacing the standard L-proline in a linear hexapeptide aldehyde series, the IC50 against replicon subgenomic RNA accumulation drops from 390 nM to 48 nM while microsomal stability in human liver microsomes (HLM, 0.5 mg/mL, NADPH regenerating system) extends the T1/2 from 11 min to 34 min. Process synthetic chemists implementing this transformation routinely couple the Boc-protected acid to the P1–P1′ prime fragment in anhydrous tetrahydrofuran using propylphosphonic anhydride (T3P®, 50 wt% in ethyl acetate, 1.8 equiv) and N-methylmorpholine (3.6 equiv) at −10 °C, quenched with 0.5 M citric acid to preserve the aldehyde function.Scaling this step in a cGMP pilot plant (reactor volume 100 L, Hastelloy C-22) specified a maximum agitation rate of 120 rpm during the exothermic activation phase; the temperature excursion exceeded the −8 °C setpoint within 90 s on the initial qualification batch, corrected by stepwise addition of T3P over 45 min using a peristaltic pump calibrated to 35.0 ± 0.5 mL/min. Residual Boc-protected amino acid was tracked by inline FTIR monitoring the carbonyl stretching band at 1698 cm−1; the endpoint criterion was <0.5 area% relative to the product peak. Analysis of the final inhibitor by LC-MS (C18, 100 mm × 2.1 mm, 1.7 µm) confirmed single-isomer purity with no detectable enantiomeric erosion, meeting the ICH Q3A qualification threshold of ≤0.10% for unspecified impurities.---Boc-cleavage streams as a feed source for macrocyclic tetrapeptide active estersRather than isolating the free amino acid after Boc removal, some CMC-driven routes channel the TFA deprotection stream directly into a reductive amination or an active ester formation. In the synthesis of an orally available thrombin inhibitor incorporating a trans-4-methyl-D-proline residue, the crude ammonium trifluoroacetate salt is taken up in dichloromethane and treated with 1.15 equiv of N-hydroxysuccinimide and 1.25 equiv of dicyclohexylcarbodiimide at 0–5 °C for 8 h. Precipitated dicyclohexylurea is removed by dead-end filtration through a 0.5 µm PTFE membrane under 0.2 MPa nitrogen overpressure; the filtrate is washed with ice-cold 5% sodium bicarbonate to extract residual TFA and dicyclohexylamine by-products. This activated ester is then added in 1.05-fold molar excess to a protected tripeptide amine fragment dissolved in dimethylacetamide, yielding the macrocyclisation precursor with 92% solution yield over three operations. In-process checks by 1H NMR (CDCl3, 400 MHz) of the succinimidyl ester must show complete disappearance of the carboxylic acid proton at δ 10.2 ppm and no residual DCC carbonyl signal at 211 ppm in the 13C spectrum before the coupling stage is initiated. Any deviation triggers a re-charge of DCC (0.3 equiv) and fresh NHS (0.25 equiv), monitored over an additional 2 h period.---In preparing a kilogram-scale shipment of the title compound for this specific application, the supplier’s quality unit performs differential scanning calorimetry analysis (DSC, Al crucible, 10 K/min) to characterise the melt endotherm and ensure no decomposition event overlaps with the shipping and warehousing temperature window. Typical onset is observed at 122–127 °C followed by an exothermic Boc-cleavage peak above 140 °C; if the exotherm shifts below 135 °C, a Lot Failure Investigation is triggered per ICH Q7 Section 2.3 to rule out contamination by acidic residues. The certificate of analysis further reports heavy metals by ICP-MS (Pb, Cd, As, Hg each <1 ppm) versus ICH Q3D Guideline Option 2B limits for oral drug products, and endotoxin levels <0.25 EU/mg when the block is destined for a sterile injectable peptide manufacture, as per Ph. Eur. 2.6.14 (Method C).---What operational boundaries govern its use as a precursor to a chiral auxiliary for phase-transfer alkylations?Deprotection of the Boc group and subsequent quaternisation of the amine yields spirocyclic ammonium salts that act as efficient chiral catalysts for the enantioselective alkylation of glycine Schiff bases. Starting from (2R,4R)-1-tert-butoxycarbonyl-4-methyl-pyrrolidine-2-carboxylic acid, the carboxylic acid is first reduced to the corresponding alcohol with borane–tetrahydrofuran complex (1.0 M in THF, 2.5 equiv) at 0 °C, followed by tosylation using 1.3 equiv of p-toluenesulfonyl chloride and 2.5 equiv of pyridine. The pyrrolidine nitrogen is then liberated via TFA, and subsequent treatment with 1.8 equiv of 3,4,5-trifluorobenzyl bromide under Schotten–Baumann conditions furnishes the chiral quaternary ammonium salt. In benzylation of the benzophenone imine of glycine tert-butyl ester with benzyl bromide under 10 mol% catalyst loading, the product is obtained in 93% ee as determined by chiral HPLC (Chiralcel® OD-H, hexane/2-propanol 99:1, 0.8 mL/min). The stereochemical outcome critically depends on rigorous exclusion of potassium carbonate hydrate contamination; the base must be dried at 200 °C for 24 h and ground to a particle size D90 < 10 µm by air-jet milling prior to use. Even trace moisture levels above 100 ppm (Karl Fischer) erode enantioselectivity to below 80% ee due to competitive non-ion-paired alkylation pathways.The sensitivity of this transformation places strict requirements on the Boc-protected precursor: residual isobutylene oligomers generated during shipping-accelerated Boc decomposition must be removed by column chromatography (silica gel, 230–400 mesh, ethyl acetate/heptane 1:3) immediately before use, as they act as phase-transfer poisons occupying the catalyst active site. A plant-scale batch designed for this route therefore implements an on-site prep-scale flash system (Biotage® Isolera LS, 1.5 kg silica cartridge) capable of processing 500 g batches in under 90 min. Fraction pooling is guided by UV threshold (254 nm, 50 mAU cutoff), cutting the main peak symmetrically to exclude the leading isobutylene-enriched shoulder confirmed by GC-headspace analysis.---The compound further finds application as a structurally defined proline surrogate in the construction of dipeptidyl peptidase-4 (DPP-4) inhibitor scaffolds where pyrrolidine ring substitution profoundly affects selectivity over DPP-8/DPP-9. Coupling the readily accessible active ester to a cyanopyrrolidine derivative in ethyl acetate at 40 °C with continuous sonication (40 kHz, 200 W) compresses the condensation time to 50 min compared to 16 h under standard magnetic stirring, with the isolated yield improving from 68% to 84% at 100 g scale. Subsequent Boc removal and final elaboration produce a candidate whose DPP-4 IC50 of 2.1 nM exceeds DPP-8 selectivity by a factor of 12,500-fold. Analytical control of residual (2R,4R) starting acid in the final active pharmaceutical ingredient is enforced via a dedicated LC-MS method (MRM transition 232.1 → 130.1) with a lower limit of quantification of 0.05 ng/mL, verifying clearance to <1 ppm in the formulated tablet.---
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The compound (2R,4R)-1-tert-butoxycarbonyl-4-methylpyrrolidine-2-carboxylic acid, CAS 169003-63-6, furnishes a stereochemically rigid proline surrogate in which a methyl substituent occupies the cis orientation relative to the carboxylic acid on the D-proline scaffold. This building block is deployed primarily as a conformational probe in the synthesis of peptidomimetic inhibitors, cyclic peptide hormones, and β-turn mimics, where the combination of ring constraint and backbone stereochemistry alters the amide cis/trans equilibrium and the φ/ψ torsional landscape. The acid-labile Boc group permits orthogonal Nα-protection strategies during solid-phase assembly using Fmoc chemistry, provided deprotection steps are conducted after completion of chain elongation.
Material destined for use as a regulatory starting material in early-phase active pharmaceutical ingredient (API) synthesis is routinely controlled against the following acceptance profile: achiral purity by HPLC-UV at 205 nm (C18 column, gradient of acetonitrile in 0.1% trifluoroacetic acid) ≥ 98.0 area%; any single unknown impurity ≤ 1.0 area%; enantiomeric excess by chiral HPLC on a Chiralpak IA-3 column (hexane/ethanol/trifluoroacetic acid 90:10:0.1 v/v/v) ≥ 99.0%; water content by Karl Fischer titration ≤ 0.5% w/w; and optical rotation [α]D20 in methanol (c = 1.0) within the range −60° to −70°. Residual solvents are quantified by headspace GC per USP ⟨467⟩ procedure A, with limits for dichloromethane (≤ 600 ppm), ethyl acetate (≤ 5000 ppm), and tetrahydrofuran (≤ 720 ppm) aligned with ICH Q3C guidance. Heavier metals are screened by inductively coupled plasma mass spectrometry (ICP-MS) and consistently fall below 10 ppm for Pd, Ni, Cu, and Fe. Lot-to-lot variability in specific rotation has been recorded at ±2° across five consecutive production campaigns, attributed to slight differences in residual moisture rather than enantiopurity shifts.
Process chemists working at the 100 mmol scale report that activated ester solutions of the acid prepared with HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) and 2.0 eq of N,N-diisopropylethylamine in anhydrous DMF at 0 °C give coupling yields > 90% onto N-terminal deprotected resin-bound dipeptides provided the carboxylic acid is pre-dried over P2O5 for 12 h under vacuum. When the same reaction is performed with HBTU, racemization—monitored by Marfey’s derivative LC-MS—climbs to 3–5% of the diastereomeric (2S,4R)-epimer after a 2 h double coupling, whereas HATU keeps the epimer content below 1%. The bulkiness of the 4-methyl group slows acylation kinetics relative to Boc-D-proline: on a Wang resin loaded at 0.4 mmol/g, the second coupling cycle for a sterically demanding Leu-Xxx dipeptide required a 4 h reaction time to reach >99% Kaiser-negative beads, compared to 90 min for the unsubstituted D-proline congener.
In a medicinal chemistry programme targeting the endothelin A receptor, replacement of the D-Pro residue in a cyclic pentapeptide lead with (2R,4R)-4-methyl-D-proline shifted the pA2 value by +0.7 log units in isolated rat aortic ring assays. The origin of this enhancement was traced by solution NMR (NOESY, 600 MHz) to a pronounced stabilization of a type VIa β-turn conformation that pre-organizes the pharmacophoric tryptophan side chain into the correct receptor-bound orientation. This conformational tightening is a direct consequence of the cis-disposed methyl group, which raises the energy barrier for ring pucker interconversion and biases the pyrrolidine ring towards the Cγ-endo envelope conformer, as determined by 3JHα-Hβ coupling constant analysis. In contrast, the (2S,4S)-L-enantiomer of this building block induces a left-handed polyproline II-like backbone kink in the same sequence context, reducing receptor occupancy by an order of magnitude. The availability of the (2R,4R) isomer thus enables stereospecific interrogation of conformational space without introducing heteroatoms that could create polar metabolic liabilities.
Global Boc deprotection after solid-phase assembly is typically executed with 95% aqueous trifluoroacetic acid (TFA) containing triisopropylsilane and water as scavengers. The crude peptide, after precipitation from cold diethyl ether, retains stoichiometric quantities of TFA as the trifluoroacetate salt of the N-terminal amine. This residual ion, if not exchanged to an acetate or chloride form, depresses the measured peptide content by 10–25% w/w and interferes with downstream N-terminal acylation. Ion chromatography on a Metrosep A Supp column (eluent: 3.2 mM Na2CO3/1.0 mM NaHCO3) routinely quantifies TFA levels of 0.8–1.2 equivalents per amine in the unpurified product. Conversion to the HCl salt by lyophilization from 0.01 M hydrochloric acid reduces TFA content to <0.05 eq, but partial acidolytic cleavage of the 4-methylpyrrolidine ring has been observed when the pH is held below 2.0 for more than 4 h. Consequently, a protocol employing ion-exchange resin (Dowex 1×8, acetate form) in 10% aqueous acetonitrile at 4 °C is adopted for sensitive constructs. Published mass balance studies indicate that incomplete counterion exchange can suppress the isolated yield of the subsequent N-terminal PEGylation step by as much as 15%, underscoring the analytical and process significance of trifluoroacetate removal.
Preparative HPLC purification of protected fragments incorporating (2R,4R)-1-Boc-4-methylproline presents a characteristic elution challenge. On a C18 column (250 × 21.2 mm, 5 µm) with a linear gradient of acetonitrile in water (both containing 0.1% formic acid), the Boc derivative elutes as a broad peak between 35% and 45% acetonitrile, frequently trailing into the solvent front of the next desired impurity. The addition of 2% isopropanol to the mobile phase sharpens the peak shape (asymmetry factor reduced from 1.8 to 1.1), but necessitates a post-collection evaporation step at ≤30 °C to avoid thermal deprotection. Scaling beyond 200 mg per injection on a 50 mm diameter column leads to resolution loss between the product and the des-Boc byproduct, which is generated by acid-mediated fragmentation during the run. A two-step catch-and-release strategy using a strong anion exchange cartridge (Waters Oasis MAX) after the first preparative pass has been implemented in process development laboratories to meet a final purity criterion of ≥99.0 area%.
| Parameter | Limit | Test Method |
|---|---|---|
| Assay (anhydrous, solvent-free basis) | ≥ 98.0% | HPLC-UV, 205 nm, external standard |
| Enantiomeric excess | ≥ 99.0% | Chiral HPLC, Chiralpak IA-3, hexane/EtOH/TFA |
| Water (Karl Fischer) | ≤ 0.5% w/w | USP ⟨921⟩, Method Ia |
| Residual solvents | ICH Q3C Option 1 limits | GC-FID headspace, USP ⟨467⟩ |
| Sulfated ash | ≤ 0.1% w/w | Ph. Eur. 2.4.14 |
| Heavy metals (Pd, Ni, Cu, Fe) | ≤ 10 ppm each | ICP-MS |
| Specific optical rotation [α]D20 in MeOH | −65° ± 5° | Polarimetry, c = 1.0 |
| Identification | 1H and 13C NMR concordant with reference | 600 MHz 1H, DMSO-d6 |
Storage stability data generated under ICH Q1A conditions indicate that the solid is physically and chemically unchanged for 24 months when kept in a tightly closed high-density polyethylene container under argon at −20 ± 5 °C and protected from light. Once opened, the material should be equilibrated to ambient temperature inside a desiccator (silica gel, activated at 120 °C) before weighing, and any unused portion should be re-sealed under dry nitrogen to forestall moisture uptake, which can reach 1.2% w/w within 4 h at 65% relative humidity.
| Xxx (pyrrolidine residue) | % cis amide population |
|---|---|
| (2S)-Pro (L-proline) | 13% |
| (2S,4S)-4-methyl-L-proline (cis) | 35% |
| (2S,4R)-4-methyl-L-proline (trans) | 8% |
| (2R,4R)-4-methyl-D-proline | 32% |
| (2R,4S)-4-methyl-D-proline (trans) | 9% |
| (2R)-Pro (D-proline) | 14% |
Conformational analysis of N-acetyl methyl ester derivatives reveals that the (2R,4R) isomer populates the cis amide rotamer to a degree essentially identical to that of the (2S,4S)-L-cis isomer but with the opposite backbone chirality. This near-mirror-image conformational propensity is exploited in the design of D-amino acid-containing macrocycles where cis amide geometry is required for ring closure. When employed in a 14-membered cyclic peptidomimetic targeting integrin αvβ3, the (2R,4R) building block allowed for a single-digit nanomolar IC50 in a solid-phase fibrinogen binding assay, whereas the corresponding trans-4-methyl-D-proline analog reduced affinity by 40-fold. The steric footprint of the methyl group also retards N-terminal degradation by prolyl oligopeptidase, as shown by a half-life extension from 1.8 h to 4.2 h in rat plasma stability studies at 37 °C. These functional distinctions, documented across multiple patent families, cement the (2R,4R) configuration as a strategic tool in peptidic drug candidate optimization.
Distinction from other Boc-protected pyrrolidine-2-carboxylic acids is essential for route selection. Boc-trans-4-hydroxy-L-proline (CAS 13726-69-7) introduces hydrogen-bonding capacity and an additional site for late-stage derivatization (esterification, glycosylation) but increases aqueous solubility and metabolic vulnerability via phase II conjugation. Boc-cis-4-fluoro-L-proline (CAS 135048-92-9) delivers an electronegative substituent that alters the pucker and stabilizes the Cγ-exo conformation, a profile opposite to the methyl analog. Boc-4-oxo-L-proline (CAS 84348-37-8) provides a ketone handle for reductive amination yet renders the ring susceptible to retro-aldol ring-opening under mildly basic conditions. The (2R,4R)-4-methyl derivative thus occupies a unique physicochemical space—hydrophobic, enzymatically stable, and predisposed to all-cis amide folds—without introducing reactive functional groups. Process development teams evaluating the (2R,4R) isomer against the more common (2S,4S)-L-cis-methyl variant cite equivalent coupling efficiency but note a 20% cost premium due to the resolution step required to isolate the D-enantiomer from racemic synthesis intermediates. Published data for direct catalytic asymmetric hydrogenation routes remain limited, though a single report using a rhodium–DuPhos system at 10 bar H2 achieved 88% ee, indicating that in-house chiral separation remains the dominant industrial method.
