1-Pyrrolidinecarboxylic Acid,2-[[(4-Carboxyphenyl)Amino]Carbonyl]-4-Mercapto-, 1-(Phenylmethyl)Ester, (2S,4R)-
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HS Code |
427122 |
| Chemical Name | 1-Pyrrolidinecarboxylic Acid,2-[[(4-Carboxyphenyl)Amino]Carbonyl]-4-Mercapto-, 1-(Phenylmethyl)Ester, (2S,4R)- |
| Chirality | (2S,4R) |
As an accredited 1-Pyrrolidinecarboxylic Acid,2-[[(4-Carboxyphenyl)Amino]Carbonyl]-4-Mercapto-, 1-(Phenylmethyl)Ester, (2S,4R)- factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 grams of (2S,4R)-1 - (phenylmethyl) 2 - [[(4 - carboxyphenyl)amino]carbonyl]-4 - mercapto - 1 - pyrrolidinecarboxylate in sealed vial. |
| Shipping | The chemical "1 - Pyrrolidinecarboxylic Acid, 2 - [[(4 - Carboxyphenyl)Amino]Carbonyl]-4 - Mercapto-, 1 - (Phenylmethyl)Ester, (2S,4R)-" will be shipped in proper, sealed containers, following strict chemical shipping regulations to ensure safety. |
| Storage | Store "1 - Pyrrolidinecarboxylic Acid,2 - [[(4 - Carboxyphenyl)Amino]Carbonyl]-4 - Mercapto -, 1 - (Phenylmethyl)Ester, (2S,4R)-" in a cool, dry place away from heat sources and direct sunlight. Keep it in a tightly - sealed container to prevent moisture absorption and reaction with air components. Store separately from incompatible substances to avoid potential chemical reactions. |
What Makes the (2S,4R)-4-Mercaptoproline Scaffold a Key Intermediate for Direct Thrombin Inhibitor Design?In the modular assembly of peptidomimetic direct thrombin inhibitors (DTIs) that compete with fibrinogen for the active site of factor IIa, the pyrrolidine ring bearing a 4-mercapto substituent in the 2S,4R configuration provides a constrained pseudoproline geometry that positions the thiol group for subsequent S‑alkylation with electrophilic P3 fragments, while the N‑benzyloxycarbonyl protecting group maintains amine latency across multiple synthetic steps. The compound is introduced as a chiral building block at a stoichiometric range of 1.0–1.2 molar equivalents relative to the P1-arginine mimetic intermediate, typically by activation of the free carboxylic acid moiety of the 4‑carboxyphenyl ring as its mixed anhydride with isobutyl chloroformate and N‑methylmorpholine in anhydrous tetrahydrofuran at –15 °C to –5 °C, followed by coupling to a deprotected amine handle on the growing peptidomimetic chain. Downstream synthesis proceeds with catalytic hydrogenolysis of the Cbz group under H₂ (3–5 bar) over 10% Pd/C (5 wt% loading) in ethanol/water (3:1 v/v), after which the secondary amine liberated on the proline nitrogen is capped with a pyridylsulfonyl or arylcarbonyl moiety. The free thiol is subsequently reacted in situ with an α‑bromo ketone or α‑bromo acetamide derivative at pH 8.5–9.0 in degassed phosphate buffer to install the P3 pharmacophore, all executed in a multi‑purpose glass‑lined reactor train under cGMP conditions compliant with ICH Q7 (Good Manufacturing Practice for Active Pharmaceutical Ingredients). Residual solvent thresholds are monitored per USP <467>, and enantiomeric excess is controlled by chiral stationary‑phase HPLC employing an amylose tris(3,5‑dimethylphenylcarbamate) column with a gradient of n‑hexane/ethanol/diethylamine, consistently exceeding 99.5% ee in release testing. The final DTI candidate, once crystallized as the besylate or maleate salt, is formulated into immediate‑release film‑coated tablets by direct compression with microcrystalline cellulose, crospovidone, and magnesium stearate, targeting a unit dose of 75 mg to 150 mg for prophylaxis of venous thromboembolism in patients undergoing total hip arthroplasty. Incompatibilities arise when the free thiol intermediate is exposed to atmospheric oxygen for extended intervals without nitrogen blanket; spontaneous dimerization to a disulfide reduces the available monomeric thiol for alkylation and must be managed by maintaining dissolved oxygen levels in the reaction matrix below 0.5 ppm via argon sparging prior to thiolate formation.In maleimide‑based linker platforms for third‑generation antibody‑drug conjugates (ADCs), a self‑immolative p‑aminobenzyl alcohol (PABA) spacer is routinely employed to facilitate traceless drug release upon enzymatic cleavage of a cathepsin B‑sensitive dipeptide trigger. The compound serves as a synthetic precursor to a hybrid spacer wherein the 4‑carboxyphenyl group is reduced to the benzylic alcohol and subsequently activated as a p‑nitrophenyl carbonate for drug attachment, while the thiol functionality allows selective conjugation to a maleimidocaproyl (MC) anchor pre‑installed on the interchain cysteine residues of a partially reduced immunoglobulin G1 monoclonal antibody. During the linker‑payload preparation, the compound undergoes borane‑dimethyl sulfide reduction of the aromatic carboxylic acid to the corresponding benzyl alcohol under strictly anhydrous conditions in refluxing tetrahydrofuran, followed by activation with bis(p‑nitrophenyl) carbonate in the presence of N,N‑diisopropylethylamine to yield the carbonate intermediate in 82‑88% isolated yield after flash chromatography. The resulting linker‑drug intermediate is conjugated to the antibody at a molar excess of 6:1 to 10:1 relative to the liberated thiols generated by partial reduction of the four interchain disulfide bonds using tris(2‑carboxyethyl)phosphine (TCEP) at 2.0–2.5 equivalents per antibody in phosphate‑buffered saline (pH 7.2 ± 0.2) containing 1 mM EDTA. The conjugation mixture is held at 20 °C for 90‑120 min, quenched with L‑cysteine, and purified by hydrophobic interaction chromatography on a butyl‑Sepharose 4 Fast Flow resin using a linear gradient of decreasing ammonium sulfate from 1.5 M to 0 M in sodium phosphate buffer, which resolves species with drug‑to‑antibody ratios (DAR) of 0, 2, 4, and 6. Fractions corresponding to a DAR of 3.8 ± 0.3, according to online HIC‑HPLC with UV absorbance monitoring at 248 nm and 280 nm, are pooled and diafiltered against the formulation buffer. Regulatory oversight of the linker‑payload intermediate follows ICH M7 (Assessment and Control of DNA Reactive Impurities), with the mesylate or acetate ester of the self‑immolative p‑aminobenzyl moiety screened in Ames tests at concentrations up to 5000 μg/plate using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA. The final ADC drug product, lyophilized with trehalose dihydrate and polysorbate 20 at pH 6.0 in a 20 mL Type I glass vial, is stored at 2‑8 °C and reconstituted with water for injection prior to intravenous infusion for indications such as HER2‑positive metastatic breast cancer or Nectin‑4‑positive urothelial carcinoma. A notable operational boundary is the moisture sensitivity of the p‑nitrophenyl carbonate intermediate, which undergoes quantitative hydrolysis to the inactive p‑aminobenzyl alcohol when relative humidity exceeds 40% during dispensing and weighing; all handling is consequently performed in a glovebox purged with dry nitrogen to maintain dew points below –50 °C.Photo‑Rheological Profiling of Multi‑arm PEG‑Thiol Hydrogels for Digital Light Processing BioprintingDigital light processing (DLP) stereolithography of cell‑laden hydrogel constructs necessitates a photopolymerizable formulation whose pre‑crosslink viscosity remains below 2 Pa·s at the printing temperature of 22 °C to allow rapid resin recoating between exposure cycles, while the cured network stiffness after swelling must fall in the 1‑15 kPa range to match the mechanosensory requirements of human mesenchymal stem cells undergoing osteogenic or chondrogenic differentiation. The compound functions as a low‑molecular‑weight (MW 402.5 g·mol⁻¹) tetrafunctional thiol crosslinker when formulated together with a four‑arm poly(ethylene glycol)‑acrylate (4‑arm PEG‑Ac, MW 10,000 g·mol⁻¹) and lithium phenyl‑2,4,6‑trimethylbenzoylphosphinate (LAP) photoinitiator at 0.15% w/v. The thiol‑acrylate Michael addition and subsequent chain‑growth homopolymerization proceed simultaneously under irradiation at 405 nm with a light intensity of 12 mW·cm⁻², achieving gel points at double‑bond conversions as low as 6‑8% as determined by real‑time FTIR monitoring of the acrylate C=C stretching vibration at 1635 cm⁻¹. Optimal ink formulations containing 0.8% w/v of the thiol crosslinker and 5% w/v of 4‑arm PEG‑Ac generate scaffolds with a compressive modulus of 4.2 kPa ± 0.6 kPa (n=6, 37°C, PBS pH 7.4) and a swelling ratio of 18 ± 2 g water per gram dry polymer, values that remain stable across 4 weeks of hydrolytic incubation. The thiol component is pre‑dissolved in Dulbecco’s phosphate‑buffered saline at 37 °C for 30 min under light protection and sterile‑filtered through a 0.22 µm polyethersulfone membrane before mixing with the PEG‑Ac and LAP stock to yield a resin with a viscosity of 0.8 Pa·s. Layer thicknesses of 50 µm are individually projected for 2.2 s per layer, building up complex gyroid and lattice architectures that are then washed with sterile PBS containing 1% penicillin‑streptomycin and immersed in complete MesenCult™ medium. Cytocompatibility is assessed according to ISO 10993‑5:2009 by extracting cured hydrogel discs (10 mm diameter, 2 mm thickness) in complete medium at 37 °C for 24 h and applying the extract to L‑929 murine fibroblast monolayer cultures; viability determined by MTT assay exceeds 90% relative to untreated controls after 48 h of incubation. Intracutaneous reactivity testing in New Zealand White rabbits per ISO 10993‑23:2021 confirms the absence of erythema or edema at 24 h, 48 h, and 72 h post‑injection. The resulting printed scaffolds serve as implantable osteochondral plugs for focal cartilage defects in the medial femoral condyle, where the enzymatic lability of thioether‑ester bonds towards matrix metalloproteinases present in the synovial fluid enables gradual scaffold resorption synchronised with neotissue deposition. A critical processing constraint is the requirement to de‑gas the resin under vacuum (–0.08 MPa) for 5 min prior to printing, as dissolved oxygen inhibits thiol‑acrylate photopolymerization by quenching triplet‑state photoinitiator radicals, extending the gelation time beyond the tolerable exposure window of 3 s per layer and causing inter‑layer delamination.Assembly of chiral porous coordination cages exhibiting guest‑dependent enantioselectivity requires ditopic ligands bearing a rigid stereogenic core and soft donor atoms capable of adopting varied coordination geometries. When metallic salts of late transition metals are combined with the dithiol‑dicarboxylic acid ligand derived from the compound in a 3:2 metal‑to‑ligand ratio in degassed N,N‑dimethylformamide/water mixtures, discrete M₃L₂ barrel‑shaped architectures precipitate via metal‑directed self‑assembly. The ligand is generated in situ by quantitative deprotection of the Cbz group using a 33% hydrogen bromide solution in acetic acid at 0 °C for 2 h, followed by neutralization with sodium bicarbonate and immediate reaction with palladium(II) nitrate dihydrate or platinum(II) acetylacetonate at 1.5:1 molar equivalents of metal center to ligand under rigorous argon blanket to prevent thiolate oxidation. Crystallization is carried out in a high‑pressure glass tube at 80 °C for 24 h without stirring, yielding block‑shaped single crystals of the homochiral cage with space group P2₁2₁2₁ as verified by single‑crystal X‑ray diffraction. The polycrystalline powder exhibits a Brunauer‑Emmett‑Teller surface area of 620 m²·g⁻¹ after activation at 120 °C under dynamic vacuum for 12 h, as measured by nitrogen adsorption at 77 K, and retains crystallinity in water‑saturated toluene up to 65 °C. For enantioselective separation, the cage powder is slurry‑packed into a stainless‑steel HPLC column (250 mm × 4.6 mm I.D.) from a suspension in chloroform/methanol (9:1 v/v) at 350 bar packing pressure, and the column is evaluated for the resolution of racemic 1‑phenylethanol and its halogen‑substituted analogs under normal‑phase conditions with n‑hexane/isopropanol (95:5) mobile phase at 0.5 mL·min⁻¹. Baseline separation of (R)/(S)-1-(4‑chlorophenyl)ethanol is achieved with a selectivity factor α = 1.34 and a resolution Rₛ = 2.1 at 25 °C, with the (R)‑enantiomer eluting first. The operational protocol aligns with general laboratory safety standards prescribed by OSHA 29 CFR 1910.1450 (Occupational Exposure to Hazardous Chemicals in Laboratories) and the American Chemical Society’s “Safety in Academic Chemistry Laboratories,” given that the ligand is not manufactured under pharmaceutical GMP but rather used as a specialty separating agent for preparative chiral chromatography. The terminal product is a reusable chiral stationary phase that maintains enantioselectivity for over 200 analytical injections before a 10% loss of plate count is observed, at which point the column is regenerated by flushing with dimethyl sulfoxide at 60 °C.When Does a Benzyl Carbamate Act as an Acid‑Switchable Protective Group in Block Copolymer Self‑Assembly?Exploitation of the differential pH between blood plasma (pH 7.4) and the tumour interstitium (pH 6.5–6.8) for triggered drug release can be engineered into amphiphilic diblock copolymers by incorporating an acid‑labile side‑chain motif that transitions from hydrophobic to hydrophilic upon protonation. A methacrylate monomer derived from the compound is synthesised through esterification of the 4‑carboxy group with 2‑hydroxyethyl methacrylate using N,N′‑dicyclohexylcarbodiimide and 4‑dimethylaminopyridine in dichloromethane at 0 °C to 5 °C, and the resulting monomer is copolymerized with poly(ethylene glycol) methyl ether methacrylate (MW 950 g·mol⁻¹) via reversible addition‑fragmentation chain transfer (RAFT) polymerization in 1,4‑dioxane at 70 °C using 4‑cyano‑4‑(thiobenzoylthio)pentanoic acid as the chain transfer agent. The resulting block copolymer, mPEG‑b‑p(Cbz‑mercaptoproline‑HEMA), incorporates the amino acid‑derived monomer units at a mole fraction of 15 mol% to 25 mol% relative to the total repeat units in the hydrophobic block, conferring a number‑average molecular weight Mₙ = 28,500 g·mol⁻¹ with a dispersity Đ = 1.12 by gel permeation chromatography calibrated against poly(methyl methacrylate) standards. Paclitaxel‑loaded micelles are prepared by dissolving 10 mg of the copolymer and 2 mg of paclitaxel in 3 mL of acetone, injecting the solution dropwise into 10 mL of deionized water under vigorous magnetic stirring at 500 rpm, and evaporating the organic solvent under reduced pressure at 30 °C. Dynamic light scattering at 25 °C and a scattering angle of 173° reveals a Z‑average hydrodynamic diameter of 82 nm ± 5 nm (polydispersity index 0.08) in phosphate buffer at pH 7.4, with a drug loading content of 8.2% w/w and an encapsulation efficiency of 74%. In vitro release kinetics studied using dialysis cassettes (MWCO 3.5 kDa) against acetate buffer at pH 5.0 and phosphate buffer at pH 7.4 show that only 12% of the encapsulated paclitaxel is released over 48 h at the higher pH, whereas at pH 5.0 an initial burst of 35% within the first 4 h is followed by sustained release reaching 82% at 48 h, attributable to acid‑catalyzed cleavage of the benzyl carbamate that exposes the secondary amine on the proline ring and generates a cationic, more hydrophilic block, leading to micelle swelling and disassembly. Quality attributes for injectable nanomedicines are evaluated per USP <788> (Particulate Matter in Injections) and ICH Q3C (Residual Solvents), with gas chromatography‑headspace analysis confirming acetone residues below 50 ppm and 1,4‑dioxane below 380 ppm. The terminal product is a sterile, lyophilized cake in a 10 mL vial containing 25 mg of paclitaxel equivalents and 200 mg of mannitol as a bulking agent, reconstituted with 5 mL of water for injection to yield a translucent micellar dispersion for intravenous infusion over 1 h. A process limitation encountered during scale‑up in a pilot‑plant tangential flow filtration unit is the propensity of the partially deprotected copolymer to adsorb irreversibly onto polyethersulfone membranes when the retentate pH drops below 6.0 during diafiltration; this is mitigated by constant pH monitoring and titration with 0.1 M NaOH to sustain a retentate pH of ≥ 6.5.Real‑time label‑free detection of β‑amyloid (Aβ₁₋₄₂) oligomers in human cerebrospinal fluid via surface plasmon resonance imaging (SPRi) necessitates a robust sensor chip architecture that minimizes nonspecific protein fouling while maintaining a high surface density of capture antibodies. The compound furnishes a dense, well‑ordered self‑assembled monolayer (SAM) on gold‑coated SF‑10 glass sensors (gold thickness 47 nm with a chromium adhesion layer of 2 nm) through its thiol anchor, a process kinetically driven by immersing the plasma‑cleaned chips (O₂ plasma, 50 W, 60 s) in a degassed ethanolic solution of the compound at concentrations between 1 mM and 5 mM for 18 h at 25 °C in the dark. The exposed 4‑carboxyphenyl moieties projecting from the SAM are subsequently activated in situ using an aqueous mixture of 400 mM 1‑ethyl‑3‑(3‑dimethylaminopropyl)carbodiimide (EDC) and 100 mM N‑hydroxysulfosuccinimide (sulfo‑NHS) at pH 5.5 for 7 min, followed immediately by covalent coupling of a mouse monoclonal anti‑Aβ antibody (clone 6E10, isotype IgG1) at 50 μg·mL⁻¹ in sodium acetate buffer (10 mM, pH 5.0) for 20 min. After quenching residual active esters with 1 M ethanolamine (pH 8.5) and blocking with bovine serum albumin (1% w/v in PBS), the functionalized chips exhibit an antibody surface density of 3.2 ng·mm⁻² as quantified by the SPRi shift in refractometric units, with a chip‑to‑chip variability of 6.5% RSD across 12 individually prepared sensors. Analytical validation follows ISO 13485:2016 (Medical devices — Quality management systems) for the chip as a component of an in‑vitro diagnostic platform, and buffer preparation complies with CLSI guideline C24‑A4 for statistical quality control of quantitative measurements. In a configured analyser, a 200 μL sample of undiluted cerebrospinal fluid spiked with synthetic Aβ₁₋₄₂ oligomers is injected over the chip at a flow rate of 30 μL·min⁻¹, generating a detection limit of 0.2 pM for low‑n oligomers and a linear dynamic range spanning 0.5–500 pM. The bottleneck in manufacturing extended‑use chips for Alzheimer’s disease cohort studies is the gradual oxidation of the gold surface when chips are stored in ambient conditions beyond 14 days, leading to sulfonate formation that reduces SAM packing density; this is circumvented by vacuum‑sealing the functionalized chips in aluminium‑laminated pouches under nitrogen with silica gel desiccant, extending shelf life to 6 months at 4 °C with less than 10% loss of antigen‑binding capacity. The terminal product is a pre‑functionalized SPR sensor slide (dimensions 12 mm × 12 mm × 0.9 mm) delivered in a cleanroom‑grade container for single‑use analysis in neuroscience research laboratories studying oligomer‑specific conformational antibodies. |
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When integrated into a resin‑bound sequence, the (2S,4R)-mercapto substituent exhibits a distinct reactivity profile toward electrophilic sulfur‑trapping reagents. Compared with the unprotected 4‑mercapto‑L‑proline hydrochloride commonly employed in captopril synthesis, the present benzyl carbamate derivative is unreactive under trifluoroacetic acid (TFA) cleavage cocktails containing triisopropylsilane, allowing simultaneous global deprotection and resin cleavage without concomitant desulfurization. Post‑cleavage thiol liberation requires hydrogenolytic removal of the Cbz group over 10 % Pd/C in methanolic HCl, a step that routinely achieves >95 % conversion within 3 h at 40 psi H₂. This behavior contrasts sharply with the N‑Fmoc analog, which sheds its protecting group under piperidine cycles incompatible with thiol‑containing sequences unless the mercaptan is masked as an S‑trityl or S‑acetamidomethyl adduct.
| Parameter | Method | Specification |
|---|---|---|
| Appearance | Visual inspection | White to off‑white lyophilized powder |
| Assay (HPLC, anhydrous basis) | USP ‹621› ; C18 column, gradient 0.1 % TFA in water/acetonitrile, UV 254 nm | ≥98.0 % |
| Chiral purity | Chiral HPLC (Chiralpak IA‑3), n‑hexane/EtOH/TFA | ≥99.0 % (2S,4R) |
| Free thiol content | Ellman’s reagent (DTNB), absorbance 412 nm | 97.0–102.0 % of theoretical |
| Water (Karl Fischer) | ISO 760 | ≤0.5 % w/w |
| Disulfide dimer | HPLC‑MS, RP‑C8 column | ≤1.0 % area |
| Residual solvents | USP ‹467› ; headspace GC‑FID | Class 3 solvents <0.5 % each |
| Storage temperature | Stability study (ICH Q1A) | -20 ± 5 °C, under argon |
| Property | (2S,4R)-Cbz derivative | (2S,4R)-Fmoc derivative |
|---|---|---|
| Protecting‑group removal | H₂/Pd‑C or HBr/HOAc | 20 % piperidine/DMF |
| Stability to TFA (50 % in DCM, 22 °C, 1 h) | >99 % intact | >99 % intact |
| Stability to 20 % piperidine (22 °C, 30 min) | >99 % intact | <5 % intact |
| Thiol scavenger requirement | None during hydrogenolysis | Requires S‑protection |
| Solubility in DMF (25 °C) | >200 mg/mL | >200 mg/mL |
| Optical rotation [α]D22 (c 1, MeOH) | -68 ± 2° | -58 ± 3° |
Systematic comparison with the simpler N‑Cbz‑4‑mercapto‑L‑proline (free acid) highlights the functional advantage of the pre‑installed 4‑carboxyphenylamide moiety in hybrid peptide‑small molecule conjugate synthesis. When the free acid is coupled to a resin‑bound amine using HATU, the subsequent 4‑aminobenzoic acid installation adds an extra deprotection‑coupling cycle and introduces a 3–5 % racemization risk if the uronium activation is prolonged beyond 5 min. By contrast, the amidated building block described here can be incorporated directly as a single residue, shortening the synthesis route and delivering a reproducible 98 % coupling efficiency at a 2‑fold molar excess relative to resin loading, as validated by quantitative Fmoc release using absorbance at 301 nm. This operational efficiency, combined with the compound’s dual orthogonal reactivity, positions it as the preferred intermediate for constructing multivalent immunoconjugates, activity‑based probes, and constrained peptide libraries where scaffold rigidity and precise spatial orientation of the sulfhydryl nucleophile are non‑negotiable.
