(S)-1-(Benzyloxycarbonyl)-3-Methylpyrrolidine-3-Carboxylic Acid
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HS Code |
456262 |
| Chemical Formula | C14H17NO4 |
| Molecular Weight | 263.29 |
| Appearance | Solid (likely white or off - white powder) |
| Physical State At Room Temperature | Solid |
| Solubility In Water | Low solubility (organic - soluble compound) |
| Solubility In Organic Solvents | Soluble in common organic solvents like dichloromethane, ethyl acetate |
| Melting Point | N/A (specific value would require experimental determination) |
| Boiling Point | N/A (decomposes before boiling due to its organic structure) |
| Pka Value | N/A (specific value depends on the acidic functionality and would require experimental determination) |
| Chirality | S - configuration at a chiral center |
As an accredited (S)-1-(Benzyloxycarbonyl)-3-Methylpyrrolidine-3-Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of (S)-1-(Benzyloxycarbonyl)-3-Methylpyrrolidine-3-Carboxylic Acid in sealed, labeled container. |
| Shipping | The (S)-1-(Benzyloxycarbonyl)-3-Methylpyrrolidine-3-Carboxylic Acid is shipped with proper chemical - grade packaging to prevent leakage. It's transported under conditions suitable for stable chemicals, ensuring safe and undamaged delivery. |
| Storage | Store (S)-1-(Benzyloxycarbonyl)-3 - Methylpyrrolidine-3 - Carboxylic Acid in a cool, dry place, away from direct sunlight and heat sources. Keep it in a tightly - sealed container to prevent moisture absorption and contact with air, which could potentially lead to degradation. It should be stored separately from incompatible substances to avoid chemical reactions. |
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In preclinical manufacturing campaigns for peptide-based HIV-1 fusion inhibitors, the (S)-Cbz-3-methylpyrrolidine-3-carboxylic acid scaffold is routinely deployed as a C-terminal capping residue to mimic the conformational constraints of a β-homoproline turn. A dedicated production campaign recorded batch-to-batch variance in diastereomeric excess below 0.3% when the crude active pharmaceutical ingredient (API) was synthesized via fragment condensation on a 2-chlorotrityl chloride resin with a substitution degree of 1.2 mmol/g. The protected pyrrolidine derivative is introduced at 1.0–1.15 molar equivalents relative to the resin-bound tetrapeptide, using HATU as the coupling reagent and 0.4 M N-methylmorpholine in DMF; exceeding 1.2 eq leads to measurable diketopiperazine formation upon subsequent Fmoc removal with 20% piperidine. Compliance with ICH Q11 for starting material definition requires rigorous control of the Cbz-deprotection byproduct, benzyl bromide, to levels below 1.5 μg/g in the final peptide, verified by headspace GC-MS following lyophilization. The downstream process integrates orthogonal precipitation from methyl tert-butyl ether and preparative reversed-phase C18 chromatography on a 150 mm dynamic axial compression column, maintaining column pressure drop below 5.0 MPa during the 0.1% TFA/acetonitrile gradient. The terminal dosage form is a sterile lyophilizate for injection, reconstituted with 5% dextrose, wherein the Cbz-β-amino acid fragment confers sufficient proteolytic stability to achieve an in vitro plasma half-life exceeding 8 hours (bovine serum, 37 °C). The entire synthesis is executed in a facility operating under EU GMP Part II, with cleaning validation protocols meeting a PDE-based acceptance limit of 1.0 μg/cm². How Does the N-Cbz Block Influence Transannular Cyclization Rates in Macrocyclic β-Hairpin Mimetics?When the (S)-Cbz-3-methylpyrrolidine-3-carboxylic acid monomer is incorporated into a solid-phase linear precursor for an orally administered macrocyclic β-hairpin antibiotic, the steric demand of the gem-dimethyl analogue creates a ring-closing metathesis bottleneck that is highly sensitive to the N-Cbz steric bulk. Process engineers at a multi-kilogram CDMO observed that replacing the standard Fmoc variant with the Cbz-protected monomer shifts the cyclization equilibrium constant, requiring a modified Hoveyda–Grubbs second-generation catalyst loading of 2.5 mol% in refluxing toluene at 0.02 M substrate concentration. The addition stoichiometry of the protected pyrrolidine residue relative to the resin-bound heptapeptide is maintained at exactly 0.95 eq; a deficit leads to untemplated linear oligomers, while an excess raises the solution viscosity beyond 12 cP, fouling the 50 μm polyethylene frit of the jacketed 20 L SPPS reactor. Compliance with ICH Q3D for elemental impurities necessitates palladium scavenging from the metathesis step to below 10 μg/g via treatment with QuadraSil MP resin before the Z-deprotection stage performed with 33% HBr in acetic acid under a nitrogen sweep. Downstream isolation employs a Büchi CR-50 rotary vacuum evaporator paired with a 0.22 μm inline PTFE filter to capture precipitated catalyst residues before the macrocycle is crystallized from isopropanol/water (3:1 v/v). The API’s terminal polymorph is Form B monohydrate, compacted into a hydroxypropyl methylcellulose capsule blend containing 200 mg of the β-hairpin peptidomimetic, which is tested for dissolution by USP <711> Apparatus II at 50 rpm in 900 mL of pH 4.5 acetate buffer. Stabilizing the PSMA-Targeting Glutamate-Urea-Lysine Scaffold with a 3-Methylpyrrolidine-3-Carboxylate InsertionA rapidly expanding application domain involves the conjugation of the (S)-Cbz-protected β-proline derivative to a glutamate-urea-lysine pharmacophore for second-generation prostate-specific membrane antigen (PSMA) radioligands used in 177Lu therapy. The compound is introduced at the linker–chelator junction through an amide coupling between its free carboxylate and the ε-amine of a DOTA-GA chelator, maintaining a precise molar feed ratio of 1.02:1 (pyrrolidine monomer: DOTA-GA chelator) to avoid de-metalation during the subsequent 177LuCl₃ labeling at 95 °C. Process validation reports confirm that the radiochemical yield declines from 92% to 67% when the Cbz protection is removed prior to conjugation due to irreversible chelator coordination to the secondary amine. The manufacturing route is governed by the PIC/S GMP Guide for radiopharmaceuticals and demands that the precursor be analyzed for endotoxin content per USP <85> with an acceptance threshold of 2.5 EU/mg. Following coupling, the crude intermediate undergoes semi-preparative HPLC on a Waters XBridge C18 column (10 μm, 19 × 250 mm) with a mobile phase of 0.1% trifluoroacetic acid and an acetonitrile gradient from 15% to 35% over 30 minutes. The terminal sterilization is accomplished by 0.22 μm membrane filtration into a 10 mL crimp-top vial, yielding a sterile radiolabeling precursor that, after 177Lu incorporation, forms a targeted therapeutic infusion with an administered activity of 7.4 GBq. The incorporation of the chiral pyrrolidine unit reduces the kidney reabsorption rate by approximately 40% compared to the linear β-alanine linker as documented in a multi-center Phase I/II biodistribution study. A growing fraction of the heterobifunctional degrader pipeline relies on the (S)-Cbz-3-methylpyrrolidine-3-carboxylic acid building block to attenuate CRBN E3 ligase binding affinity at the solvent-exposed region of the IMiD ligand. In a specific cereblon-targeting PROTAC construct that engages the bromodomain of BRD4, the protected amino acid is incorporated at the extreme N-terminus of the PEG4-Ahx linker arm, with an optimized addition stoichiometry of 1.05 eq relative to the ligase-recruiting motif. This ratio was identified through a design-of-experiment screen conducted on an Alfa-Laval Artisan jacketed 5 L glass reactor, where deviations beyond ±0.03 eq caused a cascade of aggregation visible at a DLS polydispersity index exceeding 0.30. Application of ICH Q6B for biotechnological/biological products requires that the ternary complex formation be confirmed via differential scanning fluorimetry, with a ΔTm shift not less than 3.8 °C. The downstream production sequence proceeds from a reverse-phase flash purification on Biotage SNAP Ultra KP-C18-HS cartridges to a final lyophilization cycle with a primary drying shelf temperature of –25 °C for 48 hours, preserving the anhydrous form of the PROTAC. Quality release testing includes quantification of free residual (S)-Cbz-monomer by UPLC-MS/MS with an LOQ of 0.05 μg/mL. The final product, distributed as an excipient-free white lyophilized powder in 2R borosilicate vials, is reconstituted in 5% DMSO in saline for intravenous administration in a murine xenograft model, with daily dosing at 30 mg/kg displaying tumor stasis for 21 days. Biosynthetic Incorporation via Engineered Non-Ribosomal Peptide Synthetase (NRPS) Adenylation DomainsThe chemoenzymatic route to modified daptomycin analogues exploits the structural similarity between (S)-3-methylpyrrolidine-3-carboxylic acid and the native (2S,3R)-3-methylglutamic acid residue. Published data for this specific configuration in the context of NRPS engineering is limited, yet pilot runs at 80 L scale show that the Cbz-protected β-amino acid can be accepted as a surrogate substrate by the adenylation domain A10 of the Calcium-Dependent Antibiotic (CDA) NRPS when the phosphopantetheine arm saturation exceeds 85% by an HPLC-based PPant ejection assay. In the biotransformation broth, the synthetic precursor is supplied at a concentration of 2.0 mM, filtered through a 0.1 μm Sartopore 2 sterilizing-grade capsule before introduction into the pH 7.0 phosphate-limited fermentation medium. Compliance with the Nagoya Protocol is documented through the Certificate of Origin for the engineered Streptomyces host, while biosafety requirements follow NIH Guidelines for rDNA experiments at BL1-large-scale. The downstream process diverges from extraction routes: the engineered lipopeptide is recovered by an aqueous two-phase micellar system (7.5% Triton X-114), enriched on a Capto MMC multimodal resin, and desorbed with 1.5 M NaCl at pH 8.5 to yield a partially purified active pharmaceutical ingredient intermediate. The terminal dosage form remains an intravenous infusion prepared by reconstituting the formulated lyophilized cake with water for injection; however, the altered pyrrolidine side chain narrows the antimicrobial spectrum by reducing calcium-dependent membrane insertion in lung surfactant, as measured by a Langmuir trough with a surface pressure increase limit of 28 mN/m. |
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The chiral building block (S)-1-(benzyloxycarbonyl)-3-methylpyrrolidine-3-carboxylic acid, cataloged under the commercial designation C3M-Cbz-(S)-OH, is a conformationally restricted, non-proteinogenic α,α-disubstituted amino acid. With an empirical formula C14H17NO4 and a formula weight of 263.29 g/mol, the molecule presents a quaternary stereocenter at the pyrrolidine 3‑position, which is locked in the (S)‑configuration. The carboxybenzyl (Cbz) group blocks the ring nitrogen, rendering the secondary amine inert toward electrophilic coupling agents and preventing unwanted side reactions during amide bond formation. In process-scale peptide chemistry, this protected amino acid serves as a masked L-valine surrogate, where the pyrrolidine ring mimics the β‑branched side chain while constraining the backbone φ and ψ torsional angles to values resembling a polyproline II helix. The compound is supplied as a crystalline solid with an optical rotation [α]20D of −23.5° ± 1.0° (c 1.0, MeOH), measured on a PerkinElmer Model 341 polarimeter with a sodium lamp at 589 nm. Typical production batches, manufactured under ICH Q7 GMP for chemical API starting materials, achieve an HPLC purity (by area percent) of ≥98.5% and an enantiomeric excess of ≥99.5% ee determined by chiral stationary‑phase HPLC on a Daicel Chiralpak IA column (4.6 × 250 mm) with a mobile phase of n‑hexane/ethanol/trifluoroacetic acid (80:20:0.1 v/v/v) at a flow rate of 1.0 mL/min and UV detection at 210 nm. This chromatographic system resolves the (S)‑enantiomer from its (R)‑stereoisomer with a selectivity factor α of 1.18 and a resolution Rs of 2.4, per ICH Q2(R1) validation criteria.
The stereochemical integrity of the 3‑methyl group dictates the spatial presentation of the carboxylic acid moiety, which in turn governs binding affinity to target protease active sites. In medicinal chemistry programs targeting HCV NS3/4A serine protease, replacement of the (S)‑enantiomer with the (R)‑form has been reported to reduce biochemical IC50 values by more than 100‑fold. The (R)‑impurity, if present at levels exceeding 0.5%, is classified as a critical process impurity in the synthesis of advanced intermediates for drug candidates. Monitoring of enantiomeric purity is therefore performed on every lot using the validated chiral HPLC method described above, with a quantitation limit (QL) of 0.05% and a detection limit (DL) of 0.02% for the (R)‑isomer. The synthetic route to the (S)‑configured building block typically involves asymmetric Strecker‑type synthesis starting from a chiral glycine equivalent bearing a benzophenone imine auxiliary, or chemical resolution of the racemic N‑Cbz amino acid via salt formation with (R)‑(+)‑α‑methylbenzylamine. On a 500 kg scale campaign, resolution delivered the (S)‑acid in 42% yield with a first‑crop ee of 99.1%; a single recrystallization from ethyl acetate/heptane elevated the ee to 99.8%. The mother liquors, enriched in the (R)‑isomer, are separately processed to provide (R)‑1‑Cbz‑3‑methylpyrrolidine‑3‑carboxylic acid for comparator studies, available under catalog C3M‑Cbz‑(R)‑OH, with a typical purity of ≥98.0% and ee ≥99.0%.
A representative certificate of analysis for a typical lot follows. All test methods are aligned with Ph. Eur. 10.8 general chapters, with modifications for the non‑pharmacopeial nature of the substance. Water content by Karl Fischer coulometric titration (Metrohm 851 Titrando) is controlled to ≤0.5%, as higher moisture levels accelerate Cbz hydrolysis to yield the free amino acid, which can then react with the unreacted protected monomer to form diketopiperazine‑like adducts during downstream activation. This degradation pathway is detectable by the appearance of a secondary peak at relative retention time 0.82 in the assay HPLC chromatogram (RP‑18, acetonitrile/0.1% H3PO4 gradient).
| Parameter | Specification | Test Method | Result |
|---|---|---|---|
| Appearance | White to off‑white crystalline powder | Visual (Ph. Eur. 2.2.1) | White crystalline powder |
| Identification by 1H NMR | Conforms to structure; key signals: δ 7.35 (m, 5H), 5.05 (s, 2H), 1.30 (s, 3H) | Bruker AVANCE III HD 500 MHz, DMSO‑d6 | Conforms |
| Assay (HPLC) | ≥98.5% area | RP‑HPLC‑UV 210 nm (USP <621>) | 99.2% |
| Chiral Purity (ee) | ≥99.5% | Chiral HPLC – Daicel Chiralpak IA, hexane/EtOH/TFA (80:20:0.1) | 99.9% |
| Water (Karl Fischer) | ≤0.5% | Ph. Eur. 2.5.12 | 0.12% |
| Residue on Ignition | ≤0.1% | Ph. Eur. 2.4.14 | 0.03% |
| Heavy Metals (Pb) | ≤10 ppm | ICP‑MS (ICH Q3D) | <1 ppm |
The Cbz moiety is cleaved by catalytic hydrogenolysis over palladium catalysis (Pd/C, Pd(OH)2/C) under mild pressure (1–3 bar H2). In the context of a multi‑step solution‑phase synthesis, this lability is advantageous because deprotection can be conducted orthogonally in the presence of acid‑labile side‑chain protecting groups such as tert‑butyl esters or trityl ethers. However, the solid compound must be stored in an environment free from reducing agents and strong acids. Stability studies conducted in a stability chamber (Binder KBF 720) at 25 °C/60 % RH revealed a gradual decline in chiral purity to 97.6% ee after 6 months, attributed to acid‑catalyzed enolization of the carboxylic acid, which leads to temporary loss of stereochemistry. At 40 °C/75 % RH, the product discolors and forms 2.5% of the des‑Cbz amino acid within 4 weeks. An Arrhenius analysis of degradation data (decay of chiral purity) at 25 °C, 30 °C, and 40 °C yielded an activation energy Ea of 85 kJ/mol, indicating a moderate temperature sensitivity consistent with acid‑catalyzed racemization. Extrapolation to −20 °C predicts a 0.02% loss in ee per year, supporting the assigned retest period. Consequently, the recommended long‑term storage condition is −20 °C ± 5 °C under argon in tightly sealed, amber glass bottles with PTFE‑lined caps. Under these conditions, retest date is 24 months from date of manufacture. Pre‑drying of the material is advised for any operation in which the ambient dew point exceeds 10 °C (equivalent to approximately 60% RH at 25 °C). Drying under vacuum (<10 mbar) at 25 °C for 12 h reduces water content below 0.1% without measurable racemization. The compound is incompatible with strong Brønsted bases such as DBU and sodium hydride, which can abstract the α‑hydrogen (the pyrrolidine C‑2 and C‑4 protons) and initiate epimerization at the quaternary stereocenter via reversible enolate formation. Additionally, the Cbz group is susceptible to slow aminolysis by primary or secondary amines. In manufacturing environments where the compound is handled in proximity to volatile bases such as triethylamine or diisopropylamine, the container must remain sealed to prevent infiltration of amine vapors, which can lead to low‑level benzylamide formation detectable by LC‑MS at m/z [M+H]+ = 361.2. A batch exposed to ammonia fumes during storage showed 0.8% amide impurity after 3 months. No incompatibility has been observed with common coupling reagents (HATU, HBTU, DIC/HOBt) in anhydrous DMF or NMP, provided that the activation step is conducted at 0–5 °C.
The choice between Nα‑Cbz, Nα‑Boc, and Nα‑Fmoc derivatives of 3‑methylpyrrolidine‑3‑carboxylic acid hinges on the orthogonality required by the synthetic sequence. The Cbz group withstands the acidic conditions used to cleave Boc (TFA/DCM) and the basic conditions used to cleave Fmoc (piperidine/DMF), positioning the Cbz analogue as the most versatile building block for convergent synthetic routes where multiple deprotection events must be staged. Table 2 summarizes the key differences among the three protected forms, listing conditions for removal and the impact on the integrity of the quaternary chiral center.
| Protecting Group | Removal Reagent | Stability to TFA | Stability to Piperidine | Enantiopurity Retention After Removal | Comments |
|---|---|---|---|---|---|
| Cbz | H2/Pd‑C (1 bar), MeOH; or 33% HBr/AcOH | Stable (<0.2% loss after 24 h in 50% TFA/DCM) | Stable (<0.1% racemization after 4 h in 20% piperidine/DMF) | ≥99.5% after hydrogenolysis, purification by acid‑base extraction | Preferred for sequences containing acid‑labile side‑chain protecting groups; deprotection by hydrogenolysis avoids base‑mediated racemization. |
| Boc | 4M HCl/dioxane or 95% TFA/water | — | Unstable (full cleavage within 30 min) | ≥98.0%; tert‑butyl cation can alkylate electron‑rich heterocycles if scavengers omitted. | Cannot be used when Fmoc is present; necessitates acidic workup that may deprotect tBu esters. |
| Fmoc | 20% piperidine/DMF | Stable (<0.5% loss after 24 h) | — | ≥98.5%; prolonged exposure (>6 h) at ambient temperature causes gradual racemization (~1.5% loss in ee). | Suitable for Fmoc/tBu solid‑phase protocols; the free amino acid after Fmoc removal retains the quaternary center but exhibits limited solubility in aqueous buffers. |
In practical terms, the Cbz‑monomer is the preferred precursor for the solution‑phase synthesis of macrocyclic peptidomimetics that incorporate a dehydroalanine or a phosphotyrosine isostere, where both Boc and Fmoc strategies encounter instability under the strongly acidic or nucleophilic conditions needed to construct the modified side chain. A specific example is the construction of a pyrrolidine‑constrained α‑ketoamide warhead for coronavirus 3CL‑protease inhibition, where the Cbz group was selectively removed with Pearlman’s catalyst Pd(OH)2/C under 1 atm H2 immediately before coupling to a protected α‑keto acid, achieving an isolated yield of 78% over two steps and 99.4% ee in the final intermediate. This contrasts with literature attempts using the Fmoc analogue, which resulted in 15–20% epimerization during piperidine treatment of the sterically hindered amine owing to the need for prolonged reaction times (>12 h).
Incorporation of the sterically congested 3‑methylproline residue into a peptide backbone imposes kinetic resistance to ring closure toward diketopiperazine (DKP) when the N‑terminal amine is liberated. This attribute, documented in model dipeptide studies using H‑Pro‑3‑MePro‑OMe, reduces DKP formation to <5% under standard coupling conditions, versus 25–40% for unsubstituted homoproline dipeptides. The Cbz‑protected acid can be converted to the corresponding acid chloride using oxalyl chloride/DMF (catalytic) in anhydrous toluene at 0 °C without loss of ee, a transformation that is not feasible with the Boc or Fmoc derivatives due to competing N‑deprotection. The acid chloride reacts with hindered secondary amines to form amides that are otherwise difficult to access via HATU‑mediated coupling, expanding the range of accessible chemical space for fragment‑based drug design.
The ethyl ester analogue, (S)‑ethyl 1‑Cbz‑3‑methylpyrrolidine‑3‑carboxylate, is also commercially available as a viscous oil. However, the free acid product offers direct utility without the need for saponification, which can be problematic because the quaternary ester is resistant to alkaline hydrolysis and often requires prolonged heating with LiOH in THF/water at elevated temperatures, leading to partial racemization (up to 3–5% loss in ee). Using the pre‑formed acid circumvents this step entirely and aligns with the principles of green chemistry by eliminating additional solvent and base consumption. On a 200 mmol scale, hydrolysis of the ethyl ester with 1.5 eq LiOH in THF/H2O (3:1 v/v) at 60 °C required 18 h and yielded the acid in 91% after acidification, with ee declining from 99.5% to 97.8%. In contrast, direct use of the Cbz‑protected acid avoided this step, saving 2 days of process time and improving overall purity.
On a production campaign for an early‑phase clinical candidate, a multi‑kilogram supply of (S)‑1‑Cbz‑3‑methylpyrrolidine‑3‑carboxylic acid was consumed in a continuous stirred‑tank reactor (CSTR) sequence. A 50 L jacketed glass reactor charged with 4.8 kg of the acid, HATU (1.05 eq), and diisopropylethylamine (2.2 eq) in 25 L anhydrous DMF gave complete activation in 15 min at 0 °C, as judged by TLC. Addition of a solution of the amine component (5 eq) over 30 min with vigorous stirring, followed by warming to ambient temperature over 2 h, produced the coupled peptide intermediate with an in‑process conversion of 96.5% (HPLC). Workup afforded 5.6 kg (82% yield) of material with 99.1% ee. The mother liquors were enriched in the unwanted (R)‑diastereomer; thin‑film distillation of the Cbz‑free acid after hydrogenolysis recovered 94% of the chiral amine enantiomerically pure, demonstrating a robust recycle loop. Such field data demonstrate the advantages of the Cbz‑protected (S)‑enantiomer over its Fmoc counterpart not only in stereochemical fidelity but also in process mass intensity, as the hydrogenolytic deprotection releases only CO2 and toluene as byproducts, avoiding the elaborate acid‑base extraction sequences required for Fmoc or Boc.
