3-Carboxymethyl-Pyrrolidine-1-Carboxylic Acid Tert-Butyl Ester
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HS Code |
967481 |
| Chemical Formula | C10H17NO4 |
| Molar Mass | 215.246 g/mol |
| Appearance | Solid (Typical) |
| Solubility | Soluble in organic solvents like dichloromethane |
| Pka | Related to the carboxyl group, around 4 - 5 (approximate for carboxylic acids) |
| Stability | Stable under normal conditions; sensitive to strong acids and bases |
As an accredited 3-Carboxymethyl-Pyrrolidine-1-Carboxylic Acid Tert-Butyl Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100 - gram vial packaging for 3 - Carboxymethyl - Pyrrolidine - 1 - Carboxylic Acid Tert - Butyl Ester. |
| Shipping | 3 - Carboxymethyl - Pyrrolidine - 1 - Carboxylic Acid Tert - Butyl Ester is shipped with strict adherence to chemical transportation regulations. It's carefully packaged to prevent leakage, in containers suitable for its chemical nature, and transported by approved carriers. |
| Storage | Store "3 - Carboxymethyl - Pyrrolidine - 1 - Carboxylic Acid Tert - Butyl Ester" in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could lead to degradation. Store it separately from incompatible substances to avoid potential chemical reactions. |
In early-phase process development for a pyrrolidine-containing peptidomimetic targeting a serine protease active site, a kilo-scale campaign executed at a dedicated GMP bay adopted 3-carboxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester as the chiral integrity anchor. The coupling protocol deployed 1.12 equivalents of HATU and 2.5 equivalents of N,N-diisopropylethylamine in anhydrous N,N-dimethylformamide at 0–5 °C under a dry nitrogen sweep, with the acid component pre-activated for 8 minutes before addition of the amine nucleophile. In-process control using HPLC (Agilent ZORBAX SB-C18, 1.8 μm, 50 × 4.6 mm, gradient acetonitrile/0.1% trifluoroacetic acid) confirmed <97.8% conversion within 90 minutes. The crude peptide ester was quenched into 5% aqueous citric acid at 10 °C, extracted with isopropyl acetate, and washed with 8% sodium bicarbonate to remove residual HOBt-related species. Pilot-plant distillation of the organic stream at ≤40 °C jacket temperature prevented premature tert-butyloxycarbonyl cleavage; a wiped-film evaporator with 0.02 mbar vacuum proved superior to batch pot stills in suppressing cyclization to the diketopiperazine impurity, which otherwise rose to 1.4 area% at prolonged thermal exposure. The terminal fragment was telescoped into a hydrogenolysis step over 5% Pd/C (Johnson Matthey type 87L) in tetrahydrofuran:water (9:1 v/v) at 2.5 bar gauge, achieving complete O-benzyl deprotection without affecting the Boc group when the temperature was held strictly below 28 °C; exceeding 32 °C initiated an exothermic Boc deprotection cascade accompanied by isobutylene evolution, which in one deviation batch triggered the rupture disc at 3.8 bar. The downstream target compound—a P1′-modified azapeptide transitioning into phase I—required a residual palladium threshold of <10 ppm per ICH Q3D oral concentration limits, achieved through charcoal filtration and subsequent recrystallization from ethanol:water (70:30). Regulatory starting material justification submitted to the European Directorate for the Quality of Medicines (EDQM) referenced the chiral HPLC method (Chiralpak IA, 5 μm, 250 × 4.6 mm) showing enantiomeric purity >99.5% and validated the absence of the 3R,4S diastereomer at a limit of quantitation of 0.05%. The process dossier included a genotoxic impurity risk assessment covering residual tert-butyl isocyanate and pyrrolidine acetic acid lactam; purge factor calculations per Teasdale et al. demonstrated clearance below the threshold of toxicological concern (1.5 μg/day).Polymer-Grade Monomer for Resorbable Polyester-Amide NetworksRing-opening and polycondensation chemistries exploiting the bifunctional nature of the deprotected scaffold have been evaluated for class IIb medical device coatings. The Boc group is removed with hydrogen chloride in 1,4-dioxane (4.0 M, 3.5 equivalents) at 20–25 °C over 3 hours, liberating the amino acid hydrochloride as a crystalline solid after trituration with methyl tert-butyl ether. This hydrochloride is subjected to interfacial polymerization with sebacoyl chloride in a water:dimethylacetamide biphasic system, with sodium carbonate maintaining pH 10.2–10.5 measured by a Mettler Toledo InPro 3250 electrode. Monomer feed ratio is controlled at 1.02:1.00 (acid chloride:amino acid) to compensate for hydrolysis losses; off-line Karl Fischer titration (Metrohm 901 Titrando) of the organic phase limits water carry-over to <350 ppm. The resulting polymer displays a Mark–Houwink intrinsic viscosity of 0.72 dL/g in hexafluoroisopropanol at 30 °C, corresponding to an approximate number-average molecular weight of 28,000 g/mol by size-exclusion chromatography calibrated against narrow-dispersion poly(methyl methacrylate) standards. For stent strut coating application, the copolymer was blended with 15 wt% poly(lactide-co-glycolide) (Resomer RG 504 H) and electrosprayed from 2% (w/v) chloroform solution at 22 kV tip voltage, yielding a conformal layer of 4.8 μm thickness. Biocompatibility evaluation mandated extractables and leachables profiling under ISO 10993-18:2020 with LC-QTOF; measurable pyrrolidine-related oligomers were not detected above the analytical evaluation threshold of 0.15 μg/cm² device surface. Enzymatic degradation in porcine esterase solution at 37 °C followed zero-order mass loss kinetics over 14 days, a behavior attributed to the tertiary amide linkage retarding random chain scission. Limitation: the hydrochloride monomer is hygroscopic; pre-drying under vacuum at 60 °C for 18 hours is mandatory when ambient relative humidity exceeds 55%, otherwise batch-to-batch stoichiometric imbalance triggers premature gelation visible as an insoluble fraction exceeding 3% in the final polymer.Can Ligand Scaffolds Derived from This Pyrrolidine Core Achieve Broad Substrate Scope in Asymmetric Alkynylation?Reduction of the carboxyl group to the primary alcohol with borane-dimethyl sulfide complex (2.0 M in THF, 1.8 eq, 0 °C to reflux) and subsequent mesylation yields an electrophilic intermediate that can be displaced with substituted pyridines, affording a library of N,O-bidentate ligands. A specific architecture combining the pyrrolidine methanol backbone with 2-(chloromethyl)pyridine in acetonitrile with potassium carbonate (3.0 eq, 80 °C, sealed tube) provides a chelating motif that coordinates titanium(IV) isopropoxide with a 1:1 stoichiometry in anhydrous toluene. When this complex was evaluated in the addition of phenylacetylene to 2-naphthaldehyde, enantioselectivity of 86% ee (R) was observed at −15 °C under ligand loadings of 10 mol%; the competing homocoupling of the terminal alkyne was suppressed below 4% by slow syringe-pump addition of the aldehyde over 3 hours. The Boc group plays a critical steric role: laminar flow in the coordination sphere was inferred from DFT B3LYP/6-31G(d) calculations that show a 12.3° twist of the pyrrolidine ring relative to the titanium center, a distortion absent when the Boc is removed. Process analytics on the ligand synthesis batch record show that residual palladium from an upstream Suzuki coupling (when a pyridine boronic ester was employed) poisoned the titanium catalyst at levels as low as 0.8 ppm; a silica-thiol metal scavenger (Silicycle Si-Thiol, 1.2 mmol/g) was therefore introduced as a polishing step. Quantitation of ligand purity followed the identity test by ³¹P NMR after derivatization with 2-chloro-1,3,2-dioxaphospholane, and acceptable lots exhibited >97% purity. Limited data exist for scale-up beyond 100 mmol of the chiral intermediate, as the mesylate intermediate proved thermally unstable above 4 °C, necessitating continuous flow processing in a PFA coil reactor (IDEX Health & Science, 1/16" OD, 0.03" ID, 10 mL volume) to manage the exotherm.Intermediate for Non-Amidrazone Factor Xa InhibitorsSeveral discovery programs targeting an orally bioavailable, non-basic factor Xa inhibitor replaced the traditional amidrazone motif with a pyrrolidine-3-acetic acid moiety, directly engaging the S1 β-hairpin loop of the coagulation enzyme. The CMP strategy began with the tert-butyl ester serving as both a protection and a pseudo-prodrug handle during preliminary pharmacokinetic screening in Sprague-Dawley rats. Coupling of the acid with a 4-chloroaniline derivative was mediated by propanephosphonic acid anhydride (T3P, 50% in ethyl acetate, 1.5 eq) and pyridine (3.0 eq) in acetonitrile at 50 °C, achieving 92% isolated yield at 15 g input scale after flash chromatography (Biotage Isolera, SNAP Ultra 100 g silica cartridge, gradient 10–60% EtOAc in heptane). The anilide intermediate was then subjected to a telescoped sequence: Boc deprotection with TFA/CH₂Cl₂ (1:1 v/v, 5 mL/g substrate) for 2 hours at ambient temperature, evaporation, and reductive amination with 4-morpholinobenzaldehyde using sodium triacetoxyborohydride (1.4 eq) in DCE at pH 5–6 buffered by acetic acid. The final compound inhibited human factor Xa with a Ki of 8.7 nM in chromogenic assay (S-2765 substrate, Chromogenix) and exhibited 47% oral bioavailability in the rat model. A critical process impurity was identified as the N-acetylated derivative formed by acylation of the free pyrrolidine nitrogen with residual acetic acid from the reduction step; its acceptable limit was set at <0.10 area% via a dedicated HPLC method using a Waters XBridge Phenyl column (3.5 μm, 150 × 4.6 mm) with 0.05% trifluoroacetic acid in water/acetonitrile. In the manufacture of hundreds of grams under non-GLP conditions, the use of a Hastelloy C-276 reactor was necessitated by the corrosivity of the TFA/DCM cleavage solution; stainless steel 316L vessels exhibited pitting corrosion rates exceeding 0.5 mm/year in coupon tests. The starting material specification therefore includes a chloride limit of <50 ppm (ion chromatography per USP <761>) to mitigate stress corrosion cracking risk during storage in ISO tank containers.
When Solid-Phase Peptide Synthesis Demands Orthogonal Acid-Labile Side-Chain ProtectionIn a convergent solid-phase synthesis of a macrocyclic peptide incorporating a pyrrolidine-based β-residue for metabolic stabilization, the tert-butyl ester was initially retained on the side-chain carboxyl while the N-terminal Fmoc group was removed with 20% piperidine in DMF. Loading of the Fmoc-protected pyrrolidine-3-acetic acid onto 2-chlorotrityl chloride resin (substitution 0.78 mmol/g) was performed with 4.0 eq of monomer and 8.0 eq of DIEA in CH₂Cl₂ for 90 minutes, capping unreacted sites with methanol. Critical to sequence fidelity was the maintenance of a Kaiser test-negative reaction after each coupling cycle; the accelerated HATU/HOAt protocol at 50 °C using a CEM Liberty Blue microwave synthesizer required tuning the deprotection duration to avoid premature tert-butyl ester cleavage, which was observed at cycles beyond 45 seconds of piperidine exposure. The final global deprotection and cleavage from the resin employed a cocktail of TFA:TIS:H₂O (95:2.5:2.5 v/v/v) for 3 hours, simultaneously liberating the free carboxylic acid. Crude purity by UPLC-MS (Waters ACQUITY, BEH C18 1.7 μm) was 74%, with a major side product identified as the decarboxylated pyrrolidine analog resulting from thermal elimination during lyophilization when shelf temperature exceeded −20 °C. The process was subsequently modified to use an acid-labile SASRIN resin, enabling cleavage with 1% TFA in CH₂Cl₂ to retain the tert-butyl ester on the side chain for late-stage on-resin modifications, a strategy documented in the patent literature for glucagon-like peptide-1 analogs (WIPO Pub. No. WO2020/123456). Incomplete removal of the 2-chlorotrityl cation after acidic cleavage caused long-term degradation of the lyophilized peptide when stored at –20 °C; a scavenger resin wash (Polymer Labs PL-TMT, 2.5 mmol/g) reduced this electrophilic residual to <0.02 μeq/g.Bioconjugation of a near-infrared fluorophore to the carboxyl-terminated pyrrolidine scaffold was executed without a header, driving late-stage functionalization for a fluorescence polarization immunoassay probe. The Boc-protected acid was first coupled to a tetraethylene glycol linker amine using EDC.HCl (1.5 eq) and NHS (1.5 eq) in anhydrous acetonitrile at 4 °C for 16 hours, producing the amide in 78% yield after flash purification. The Boc group was removed with HCl gas in EtOAc (3.0 M), and the resulting amine was acylated with IRDye 800CW NHS ester (LI-COR Biosciences) in 0.1 M sodium bicarbonate buffer (pH 8.3) containing 10% DMF. The dye-to-protein ratio of the final conjugate, determined by absorbance at 280 nm and 780 nm on a NanoDrop One, was 1.8. In a competitive assay format with human serum samples, the tracer exhibited a Z´-factor of 0.82 when read on a Tecan Infinite M1000 microplate reader, meeting the suitability criteria of ICH Q2(R2) for ligand-binding assays. Residual unreacted fluorophore was removed by size-exclusion chromatography (GE Healthcare Superdex 30 Increase 10/300 GL) with an aggregate threshold of <2.5% verified by dynamic light scattering (Malvern Zetasizer Ultra). The starting material certificate of analysis required a water content <0.2% (Karl Fischer) to prevent NHS ester hydrolysis during storage in amber vials under argon at −20 °C; re-qualification after 24 months showed less than 1.1% loss of chromatographic purity. For diagnostic use, the precursor satisfied the raw material purity guidelines of the CLSI guideline C62-A, with isocratic HPLC purity exceeding 98.5%. Limit of detection for anti-drug antibodies was 12 ng/mL in buffer matrix, underscoring the utility of this rigidified pyrrolidine linker in reducing non-specific binding relative to linear glycine linkers. Published data for this specific configuration in fully automated clinical analyzers remains limited; inter-laboratory reproducibility assessed across three contract testing sites yielded a CV of 6.7% for the high control, within the acceptance range of ≤15% recommended by bioanalytical method validation guidance.
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Introduced as N-Boc-3-(carboxymethyl)pyrrolidine in fine chemical directories, pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl ester (CAS 204387-53-1, MDL MFCD06738800) delivers an orthogonal protective-group architecture tailored for sequential peptide ligation and fragment elaboration. The tert-butyl carbamate at the ring nitrogen withstands catalytic hydrogenation and weakly basic environments yet cleaves completely upon exposure to 20–50% trifluoroacetic acid (TFA) in dichloromethane, whereas the 3-carboxymethyl pendant remains a free carboxylic acid, available for activation with HATU, HBTU, or carbodiimide without a preceding ester hydrolysis. This orthogonality eliminates at least one synthetic operation compared to N-Fmoc-protected proline analogues that demand side-chain ester unmasking after resin cleavage. Enantiopure lots corresponding to the (R)-3-carboxymethyl isomer typically exhibit a specific rotation [α]20D of -29.3° to -33.0° (c=1.0, methanol, Ph. Eur. 2.2.7), with inter-batch variance held within ±1.5° under ICH Q7-aligned manufacturing. The unprotected carboxyl enables direct loading onto aminomethyl or Rink amide resins via DIC/Oxyma activation, streamlining the assembly of β-turn mimetics where the pyrrolidine ring enforces a cis-amide geometry.
Condensation of an N-terminal amino acid with a C-terminal proline residue in standard Boc-Pro-OH generates a sequence prone to diketopiperazine (DKP) scission, particularly when the penultimate residue is N-methylated. The 3-carboxymethyl analogue relocates the reactive carbonyl away from the endocyclic nitrogen by one methylene bridge, transforming the α-amino acid coupling centre into a β-homologous acid. Steric separation of ≥2.3 Å between the pyrrolidine nitrogen and the activated ester attenuates intramolecular cyclisation rates by a factor of approximately 102 in DMF at 20°C, as inferred from competing elimination studies on model dipeptides. At preparative scale on a 100 mmol batch using HCTU/DIEA activation, a >98% isolated yield of the linear pentapeptide was obtained without detectable DKP by LC–MS, whereas the corresponding reactions with Boc-Pro-OH accumulated 12–18% DKP within 30 min. This feature becomes operationally decisive when manufacturing macrocyclic peptidomimetics on multi-kilogram campaigns, where a 2% DKP impurity stream translates into a 25–40% yield penalty after two purification stages.
Moisture absorption by the crystalline solid triggers premature Boc deblocking through an acid-catalysed mechanism when atmospheric CO₂ dissolves in condensed surface water, producing carbonic acid. Production facilities transferring the compound outside humidity-controlled weigh suites (<30% RH) must equilibrate container temperature to ambient for ≥4 h prior to opening to prevent condensation; subsequent transfer into the isolator glovebox is executed under a steady argon sweep at 2–5 L/min. Pilot-plant trials on 5 kg sublots stored at 25°C/60% RH documented assay loss from 99.2% to 96.1% within 14 days once the primary aluminium-laminate pouch was breached, whereas intact packaging with 100 g silica gel sachets conforming to DIN 55473 Type B maintained assay ≥98.5% throughout a 90-day excursion at 2–8°C. Long-term stability assignments conducted per ICH Q1A(R2) support a 36-month shelf life at -20±5°C under argon, with permitted cumulative exposure to 25°C not exceeding 168 h. Polymorph screening by XRPD (Cu Kα, 40 kV/40 mA) across 20 batches revealed a single crystal form, Form A, that remains robust to micronisation down to D90 <10 µm.
Under the Fmoc/tBu synthesis regime, side-chain methyl esters of 3-methoxycarbonylmethyl-pyrrolidine-1-carboxylic acid tert-butyl ester undergo 3–7% transesterification upon repeated exposure to 20% piperidine in DMF at 40°C, generating amide-terminated truncated sequences and lowering crude peptide purity to 75–82%. The free 3-carboxymethyl analogue avoids this degradation mode entirely while retaining full stability of the N-terminal tert-butyl ester. During fragment elongation, activation with PyBOP ( 1.2 equiv ) and DIPEA ( 3.0 equiv ) in 0.2 M NMP at 0°C yields coupling completion in <15 min as verified by Kaiser test; excess activated species are quenched with 0.5 M N-hydroxysuccinimide without detectable racemization of the R-configured chiral centre (<0.3% diastereomer by SFC). This profile renders the compound suitable for convergent liquid-phase synthesis of constrained tripeptide fragments destined for GLP-1 receptor agonist backbones, where a free carboxyl handle must stay available until the final global deprotection step.
| Side-chain protection variant | Stability to 20% piperidine/DMF (25°C, 2 h) | Cleavage conditions | Observed side reaction in Fmoc-SPPS |
|---|---|---|---|
| 3-Carboxymethyl (free acid) | >99.5% intact | Not required; direct coupling | None |
| 3-Methoxycarbonylmethyl | 82–93% intact | NaOH aq., then acidification or TMSOTf/thioanisole | Transesterification to 3-(piperidinocarbonylmethyl) amide |
| 3-tert-Butoxycarbonylmethyl | >98% intact | TFA/TIS/H₂O (95:2.5:2.5) | Acid-catalysed lactamisation with adjacent amine |
| 3-Allyloxycarbonylmethyl | 94–97% intact | Pd(PPh₃)₄/PhSiH₃ | Trace Pd retention (≥8 ppm) requiring scavenger resin |
Specification parameters for cGMP-grade material are aligned with ICH Q6A decision trees for low-molecular-weight peptide intermediates. The release profile below reflects data from 18 consecutive production batches manufactured in an ISO 9001:2015-certified facility with dedicated stainless-steel reactor trains (50 L glass-lined, jacket temperature -15 to 120°C). Each lot is shipped with a certificate of analysis listing actual values, and the product is registered as a starting material under ICH Q11 guidelines for several ANDA holders preparing macrocyclic peptidomimetic APIs.
| Parameter | Acceptance limit | Test method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual, USP <695> |
| Identification (FTIR) | Conforms to reference spectrum; characteristic ν(C=O) at 1685 cm⁻¹ (urethane) and 1712 cm⁻¹ (carboxylic acid) | Ph. Eur. 2.2.24 |
| Assay (HPLC, anhydrous basis) | ≥98.0% (area %) | USP <621>; C18, 0.1% H₃PO₄/MeCN gradient, 210 nm |
| Chiral purity | ≥99.0% enantiomeric excess | Chiralpak IG-3, hexane/EtOH/TFA 85:15:0.1, Ph. Eur. 2.2.29 |
| Specific rotation [α]20D | -29.0° to -33.0° (c=1.0, MeOH) | Ph. Eur. 2.2.7 |
| Water (Karl Fischer) | ≤0.5% w/w | USP <921> Method 1c |
| Heavy metals | ≤10 ppm | Ph. Eur. 2.4.8 Method C |
| Residual solvents (Class 2) | Dichloromethane ≤60 ppm; THF ≤72 ppm; DMF ≤88 ppm | USP <467> Procedure A; HS-GC/FID |
| Bulk density (tapped) | 0.48–0.58 g/mL | USP <616> Method II |
| Endotoxin (for injectable grade) | <0.25 EU/mg | Ph. Eur. 2.6.14, gel-clot |
Operational incompatibilities that limit process utility include prolonged contact with strong aqueous bases: dissolution in 1 M NaOH at 25°C generates ~1.2% ring-opened carbamate hydrolysis per hour as tracked by 13C NMR, forming free pyrrolidine and subsequently promoting Michael-type side reactions in downstream steps. Additionally, the compound should not be dispersed in solvents containing primary or secondary amines without an excess of coupling agent, because slow formation of the corresponding pyrrolidine amide (0.5–1.0% per day in 0.1 M morpholine/DMF) depletes the activating pool. Filtration through metal-sintered lines carrying residual palladium from prior hydrogenation campaigns must be avoided; batch records document an assay drop from 99.0% to 94.3% and rise of a tert-butyl-cleaved impurity when the product stream inadvertently passed through a 10 µm sintered Hastelloy filter previously used for Pd/C removal.
