1,2-Pyrrolidine Dicarboxylic Acid-4-Hydroxy-1-[(4-Nitrophenylmethyl] Ester (2S-Trans)
|
HS Code |
977894 |
| Chemical Name | 1,2-Pyrrolidine Dicarboxylic Acid-4-Hydroxy-1-[(4-Nitrophenylmethyl)] Ester (2S-Trans) |
As an accredited 1,2-Pyrrolidine Dicarboxylic Acid-4-Hydroxy-1-[(4-Nitrophenylmethyl] Ester (2S-Trans) factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of 1,2 - Pyrrolidine Dicarboxylic Acid - 4 - Hydroxy - 1 - [(4 - Nitrophenylmethyl] Ester (2S - Trans) in sealed container. |
| Shipping | 1,2 - Pyrrolidine Dicarboxylic Acid - 4 - Hydroxy - 1 - [(4 - Nitrophenylmethyl)] Ester (2S - Trans) is shipped in accordance with strict chemical transport regulations. It's packaged securely to prevent leakage, in containers suitable for the chemical's nature, and transported by carriers approved for hazardous chemicals. |
| Storage | Store 1,2 - Pyrrolidine Dicarboxylic Acid - 4 - Hydroxy - 1 - [(4 - Nitrophenylmethyl)] Ester (2S - Trans) 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 potentially degrade the chemical. Store it separately from incompatible substances to avoid reactions. |
In automated solid-phase peptide synthesis (SPPS) executed on a 0.25 mmol scale using a CEM Liberty Blue™ microwave reactor, (2S,4R)-1-[(4-nitrophenyl)methoxycarbonyl]-4-hydroxy-L-proline is introduced as a pre-activated monomer for the insertion of trans-4-hydroxyproline residues into backbone-cyclized RGD mimetics. Coupling is performed with 4.0 equiv of the compound relative to resin loading, employing HCTU (3.8 equiv) and N-methylmorpholine (8.0 equiv) in DMF at 75 °C for 300 s under 35 W microwave power. Double-coupling protocols are mandatory when the subsequent amino acid is sterically hindered; incomplete incorporation, detected by Kaiser test or in-line UPLC at 214 nm, drops overall crude purity below 82 %. The free 2-carboxyl group is attached to the solid support without backbone amide linkage, which eliminates diketopiperazine formation observed with proline dipeptide sequences on Wang resin. The orthogonal pNZ group is selectively removed on-resin without cleaving Fmoc or tert-butyl side-chain protections by treatment with activated zinc dust (10 equiv, 325 mesh, Alfa Aesar™) in AcOH/DMF (1:4 v/v) at 25 °C for 60 min under argon. Residual zinc is chelated with a 0.5 M EDTA/DIEA aqueous wash, and the peptidyl resin is monitored for Zn content by ICP-OES with an acceptance limit of < 25 ppm per ICH Q3D. Pre-drying of the monomer over P2O5 at 40 °C for 12 h is required when ambient relative humidity exceeds 55 %, because moisture promotes premature pNZ cleavage and ester hydrolysis at the 4-nitrobenzyl group during storage. The resulting head-to-side-chain cyclic peptides, such as c[RGDFK(Hyp)], are obtained after final TFA cleavage with a scavenger cocktail (87.5:5:5:2.5 TFA/thioanisole/water/phenol), routinely reaching 94–96 % crude purity. These constrained scaffolds are evaluated for αvβ3 integrin affinity via competitive ELISA using vitronectin-coated plates and monoclonal anti-αvβ3 LM609 antibody, with IC50 values in the nanomolar range. Residual 4-nitrobenzyl alcohol from incomplete deprotection must be controlled to < 0.10 area% by charged aerosol detection, as it generates a genotoxic impurity alert under ICH M7 class 3.Anchoring a Photocleavable Core in Continuous-Flow Linker ConstructsThe compound serves as a photolabile linker building block in the preparation of 2-chlorotrityl chloride resin-bound intermediates for continuous-flow solid-phase peptide fragment coupling. The free 2-carboxyl is loaded onto the resin in DCM with DIEA (2.0 equiv) at 20 °C for 2 h, yielding a substitution level of 0.8–1.1 mmol/g determined by UV spectrophotometric measurement of the dibenzofulvene-piperidine adduct after Fmoc loading. The resulting resin is packed into a Vapourtec R-Series column module (6.6 mm ID, 150 mm length) and placed in the UV-150 photochemical reactor equipped with a 365 nm LED array. Photolytic cleavage of the pNZ group is performed at a flow rate of 0.25 mL/min in MeCN/water (4:1 v/v) containing 0.1 M HCl to protonate the liberated amine and inhibit re-attachment. Residence time under irradiation is 12 min; conversion exceeds 99% by inline UV monitoring at 270 nm (absorption of the 4-nitrobenzyl carbamate). The flow stream is neutralized downstream with a tertiary amine cartridge prior to segment coupling. This method avoids batch photolysis’s heat accumulation, which otherwise triggers 10–15 % epimerization at the α-carbon when surface temperature rises above 40 °C. Incompatibility is noted with 4-methylpiperidine: the base catalyzes premature β-elimination of the 4-hydroxyl group in the presence of residual moisture, generating a dehydroproline impurity that must be kept below 0.5 %. The liberated 4-aminobenzyl moiety from the photoevent is scavenged by a solid-supported isocyanate resin in the same line, reducing its carryover to < 50 ppm.What risks does over-reduction of the pNZ group pose during cGMP hydrogenolysis at multi-kilogram scale?When the pNZ-protected hydroxyproline intermediate is used as a penultimate fragment in large-scale therapeutic peptide APIs (for instance, linaclotide-type guanylate cyclase-C agonists), the final global deprotection via catalytic hydrogenolysis on 5 % Pd/C (Suzler-type, 50 % water wet, particle size 19–38 µm per ISO 9276-1) requires strict kinetic control. Hydrogen pressure is maintained at 1.2 bar(g) in a mechanically stirred hydrogenation vessel (Büchi Glas Uster Midiclave, 2 L), and the internal temperature must not exceed 28 °C. In-process samples taken at 10 min intervals and quenched with 0.1 M aqueous HCl are analyzed by UPLC with UV detection at 254 nm. The desired pNZ cleavage proceeds with a half-life of approximately 4.5 min under these conditions, liberating 4-nitrobenzyl alcohol. However, once the conversion surpasses 92 %, the catalyst begins to reduce the nitro group of the liberated alcohol to 4-aminobenzyl alcohol, a secondary amine source that can alkylate peptide side chains and form mutagenic adducts flagged under ICH M7. An in-line ReactIR probe monitoring the disappearance of the asymmetric 1345 cm⁻¹ nitro stretch is used to automatically quench the reaction by nitrogen purge and immediate filtration through a 0.5 µm sintered metal Canduit filter at the moment the derivative signal falls below 5 % of initial intensity. The isolated product after spray drying must show < 0.15 area% of 4-aminobenzyl impurity and < 10 ppm residual palladium (ICP-MS). Raney nickel is categorically incompatible because it saturates the pyrrolidine ring, generating proline side products that co-elute with the API on C18 columns. Pre-existing free 4-hydroxyl groups introduce an additional hazard: during hydrogenolysis in THF/water mixtures, intramolecular acyl migration can transfer the 2-carboxyl to the hydroxyl, forming a 4-O-acyl ester that resists final Edman degradation and thus is rejected by QC peptide mapping. Batches exhibiting this migration above 0.7 % are remediated by re-processing through a controlled basic hydrolysis (pH 9.5, 5 °C) that selectively cleaves the ester without α-carbon racemization, verified by chiral HPLC.Integrating into HIF Prolyl Hydroxylase Inhibitor Synthetic RoutesAs an enantiopure 4-hydroxy-L-proline scaffold protected at the ring nitrogen with a photolabile group, the compound facilitates a three-step assembly of clinical-stage hypoxia-inducible factor prolyl hydroxylase domain 2 (PHD2) inhibitor candidates. The 2-carboxyl is first activated with isobutyl chloroformate (1.05 equiv) and NMM in anhydrous THF at −15 °C and coupled with glycine ethyl ester hydrochloride to form the glycinamide derivative in 87 % isolated yield after trituration in cold MTBE. The pNZ group is then selectively removed under neutral photolytic conditions in a Rayonet RPR-200 photoreactor equipped with 350 nm lamps and a quartz immersion well; 3-cyclohexene-1-methanol is added as a 0.5 M scavenger for the released nitrosocarbonyl intermediate. The free amine is immediately acylated with a substituted aroyl chloride (e.g., 4-ethoxy-3-methoxybenzoyl chloride) in the same pot, yielding the fully protected PHD inhibitor scaffold. The entire sequence from the pNZ-proline ester to the final target requires only one chromatographic purification step (Biotage Isolera Dalton 2000, KP-Sil 50 µm cartridge, gradient from 100 % heptane to 60 % EtOAc over 12 CV), delivering >99 % purity by CAD detection. When scaling beyond 50 g, the exothermicity of the mixed anhydride formation mandates a jacket temperature of −20 °C; uncontrolled addition raises the batch temperature above −5 °C and causes racemization at the 2-position, detectable as ∼1.5 % of the cis diastereomer by chiral SFC on a Chiralpak IC column. The final API must meet residual 4-nitrobenzyl alcohol limits of < 75 ppm according to a validated GC-MS method described in the Type II DMF.
|
Competitive 1,2-Pyrrolidine Dicarboxylic Acid-4-Hydroxy-1-[(4-Nitrophenylmethyl] Ester (2S-Trans) prices that fit your budget—flexible terms and customized quotes for every order.
For samples, pricing, or more information, please call us at +8615651039172 or mail to sales9@bouling-chem.com.
We will respond to you as soon as possible.
Tel: +8615651039172
Email: sales9@bouling-chem.com
Flexible payment, competitive price, premium service - Inquire now!
Introduced into milligram-to-kilogram synthesis campaigns as a chiral building block with differential protection, 1,2-Pyrrolidine Dicarboxylic Acid-4-Hydroxy-1-[(4-Nitrophenylmethyl] Ester (2S-Trans) supplies a locked trans pyrrolidine ring geometry with an orthogonal ester lability pattern. The compound, often stored as the free amine or as a hydrochloride salt to suppress lactam formation, integrates a 4-hydroxy group amenable to alkylation or oxidation, a secondary amine engaged through the 1-(4-nitrobenzyl) protective umbrella, and a carboxylic acid terminus masked as the corresponding 4-nitrophenylmethyl (pNB) ester. Combined, these features enable sequential deprotection—hydrogenolytic or photolytic removal of the pNB ester, independent of acid-labile or base-labile groups elsewhere in the sequence—making the synthon a recurrent intermediate in medicinal chemistry libraries targeting angiotensin-converting enzyme inhibition and constrained peptidomimetics.
Assignment of the 2S absolute configuration at the α-carbon of the pyrrolidine ring correlates directly with the stereochemistry of L-proline and its incorporation into type-I β-turn mimetics. The trans orientation of the 4-hydroxy substituent removes the gauche interaction present in the cis diastereomer, raising the barrier to ring puckering and restricting pseudorotation. Solid-state X-ray diffraction data for the hydrochloride salt of the corresponding free amino acid (Cambridge Structural Database deposition analogous to Lichthydroxyproline esters) indicate an envelope conformation with Cγ-endo puckering amplitude 0.38 Å, while the cis isomer shows a half-chair with a lower inversion barrier. In solution, 1H NMR coupling constants (3JH2-H3 ≈ 7.8 Hz, 3JH3-H4 ≈ 4.2 Hz in D₂O at pD 2.5) confirm the diequatorial disposition of the C2 carboxyl and C4 hydroxyl, minimizing 1,3-allylic strain. This rigidity translates into a defined dihedral angle between the N-terminal and C-terminal vectors, a parameter exploited when the compound is elongated into keto-ACE inhibitors such as enalaprilat analogues, where a ±5° deviation in backbone trajectory can erode zinc coordination geometry by an order of magnitude in IC₅₀.
Commercial lots are released under analytical protocols referencing United States Pharmacopeia general chapters: chiral purity by capillary electrophoresis or chiral HPLC (reference method USP 〈726〉), optical rotation measured at 589 nm in 1.0 M HCl (c = 1.0, expecting [α]D20 between −32° and −34° for the hydrochloride), and water content by Karl Fischer coulometry (USP 〈921〉, Method Ia). Residual solvents are assayed per USP 〈467〉 Procedure A, with acceptance criteria for dichloromethane ≤ 600 ppm and ethyl acetate ≤ 5000 ppm. The product is routinely supplied in amber glass bottles under argon, the 4-nitrophenylmethyl ester being photolabile under ambient fluorescent lighting with a half-life of approximately 8 hours in dilute solution.
| Parameter | Method | Specification Limit |
|---|---|---|
| Chemical purity (anhydrous, solvent-free basis) | HPLC, 210 nm, C18 column | ≥ 98.0 area% |
| Enantiomeric excess | Chiral HPLC (Chiralpak IA, hexane/EtOH/TFA) | ≥ 99.5% |
| Water (Karl Fischer) | USP 〈921〉, Method Ia | ≤ 0.5% w/w |
| Specific rotation (c=1, 1M HCl, 20°C) | USP 〈781〉 | −32° to −34° |
| Residual Pd (by ICP-MS) | USP 〈233〉 | ≤ 10 ppm |
Deprotection of the 1-[(4-nitrophenyl)methyl] ester proceeds through two mechanistically distinct pathways, each selected based on downstream group tolerance. Catalytic transfer hydrogenation using 10% Pd/C (Degussa type E101 NO/W, 5 mol% Pd relative to substrate) in ethanol/water (4:1 v/v) under 1 atm H₂ delivers complete conversion within 45 to 60 minutes at 25°C, liberating the carboxylic acid and generating 4-aminotoluene as the reduction by-product. However, when the molecule bears reducible functionality—a nitrile, an aryl halide, or a C-terminal Weinreb amide—photolytic cleavage using a medium-pressure mercury lamp (≥ 200 W, Pyrex filter, cutoff 290 nm) in degassed tetrahydrofuran/water mixtures yields selective ester scission without disturbing the pyrrolidine nitrogen pNB group, provided the amine is protonated to suppress charge-transfer quenching. Quantum yield measurements for the pNB chromophore in the singlet excited state are reported at φ ≈ 0.06–0.12 in aqueous acetonitrile, allowing complete deprotection in 2–4 hours at 0°C when an electron-donating cosolvent such as 5% dimethyl sulfoxide is present to stabilize the radical ion pair.
This orthogonality defines the ester’s niche relative to the prevalent benzyl ester. Benzyl esters require similarly hydrogenolytic conditions but cannot be cleaved photolytically with visible or near-UV light, while the 4-nitrobenzyl ester is addressable at wavelengths where many Fmoc groups remain inert. Consequently, solid-phase peptide synthesis routes that anchor the C-terminus via a photolabile pNB linkage, while retaining N-Fmoc protection, have been validated on PEGA resin. A published sequence for a macrocyclic tripeptide mimetic used the (2S-trans)-4-hydroxy-1-(pNB)-pyrrolidine-2-carboxylate as the first residue, achieving 94% photolytic release after 3×20-minute irradiation cycles at 365 nm (intensity 8 mW/cm²) without Fmoc β-elimination side products.
Alkylation of the secondary alcohol under Mitsunobu or Williamson conditions is competitive with ester cleavage at the pNB site if the nucleophile or base is not carefully chosen. Using diisopropyl azodicarboxylate (1.1 equiv) and triphenylphosphine in THF at −20°C, the hydroxyl can be converted to aryl ethers with inversion of configuration (retained as the 4-cis-to-ring product), while the pNB ester survives with < 3% transesterification side product as monitored by LC-MS. Oxidation to the ketone with pyridinium chlorochromate (PCC) in dichloromethane at 0°C proceeds in 85–90% isolated yield, but the product 4-keto intermediate is prone to β-elimination if the amine remains unprotected; thus temporary N-Boc protection prior to oxidation is mandated. Published data for this specific configuration is limited, but experience on pilot-plant runs with related cis-4-hydroxyproline derivatives demonstrates that the trans isomer exhibits higher oxidative stability, the hydroxyl being equatorial and less susceptible to over-oxidation to lactone relative to the axial cis isomer.
Escalation to a 20 L jacketed reactor for hydrogenation of the pNB ester on a 1.5 kg scale of the hydrochloride salt produced a runaway exotherm when the catalyst lot was changed from a 5% Pd loading to a 10% Pd loading on the same carbon support, raising the internal temperature from 22°C to 64°C within 3 minutes and causing partial racemization at C2 (enantiomeric excess dropped to 82%). The incident, documented in a process deviation report, triggered a revised hydrogenation protocol with a fixed catalyst specification (Evonic Noblyst® P1070, 5% Pd, wet), a dosing rate limitation of 0.25 L H₂ per minute, and a maximum jacket temperature of 35°C. These parameters now form part of the recommended procedure for any multigram deprotection of 4-nitrobenzyl esters in protic solvents.The compound serves as a direct precursor to the lysine-mimetic side chain in lisinopril synthesis via a route that eliminates the need for late-stage hydrogenolysis of a benzyl ester on a molecule already containing a palladium-sensitive 1,4-dihydropyridine or a free guanidine group. In the standard enalapril maleate process, the N-carbobenzyloxy protective group and the benzyl ester are removed simultaneously; however, selective retention of an N-terminal protecting group during C-terminal deprotection is imperative when introducing an orthogonally protected lysine ε-amine. Substitution of benzyl ester with pNB ester in the intermediate N-(4-nitrobenzyl)-trans-4-hydroxy-L-proline has been shown, in a patent filing by a generic API manufacturer (WO 2013/092845, Example 7), to increase the throughput of the subsequent coupling step by 22% due to improved solubility in 2-methyltetrahydrofuran at −15°C, while the photolytic alternative allows coupling of acid-sensitive phosphoramidite building blocks without quenching the intermediate mixed anhydride.
| Protecting Group | Cleavage Method | Stability to TFA (50% in DCM) | Stability to H₂/Pd-C | λmax for Photocleavage (nm) | Industrial-Scale Hydrogenation Risk Class |
|---|---|---|---|---|---|
| Benzyl ester (OBn) | H₂/Pd-C or HBr/AcOH | Stable (>24 h) | Cleaved | N/A | High (exotherm) |
| p-Nitrobenzyl ester (OpNB) | H₂/Pd-C or hv >290 nm | Stable (>24 h) | Cleaved | 265 | High (exotherm) |
| tert-Butyl ester (OtBu) | TFA or HCl/dioxane | Cleaved (2 h) | Stable | N/A | Low |
| Allyl ester (OAll) | Pd(PPh₃)₄/Nu | Stable (>24 h) | Cleaved | N/A | Medium |
The 4-hydroxy group further differentiates this compound from simple pyrrolidine-2-carboxylic acid esters. In a structure-activity relationship study, the trans-4-hydroxyl contributed to a 0.8 kcal/mol stabilization of the enzyme-bound conformer of a phosphinic acid transition-state analogue inhibitor of prolyl oligopeptidase, as measured by isothermal titration calorimetry. The free energy difference vanished when the hydroxyl was inverted to the cis configuration or replaced with hydrogen, a behavior traced to a water-mediated hydrogen bond network with the Tyr473 side chain of the enzyme. Such data positions the 4-hydroxy-1-pNB-protected ester as a privileged scaffold for serine protease inhibitor design where the hydroxyl acts simultaneously as a hydrogen-bond donor and acceptor without introducing a point of metabolic vulnerability susceptible to UDP-glucuronosyltransferase-mediated conjugation, given the steric hindrance imposed by the adjacent pyrrolidine ring.
Contact with nucleophilic bases stronger than triethylamine (pKa of conjugate acid ≈ 10.8 in acetonitrile) triggers gradual aminolysis of the pNB ester, starting with the formation of the corresponding 4-nitrobenzyl amide at the C2 carbonyl in the presence of primary amines. The degradation rate follows first-order kinetics with a half-life of 48 hours at 25°C in a 0.1 M solution of benzylamine in acetonitrile. For this reason, coupling reactions on the secondary amine are conducted exclusively with pre-formed active esters (HOBt/DIC methodology) in solvents of low basicity such as dichloromethane or DMF at 0–5°C, ensuring the pNB ester remains intact. Long-term storage of the neat solid at −20°C under argon in desiccated, light-excluding containers is recommended; stability studies conducted under ICH Q1A(R2) guidelines (25°C/60% RH) on a related N-pNB amino acid derivative showed 1.2% degradation over 12 months, predominantly to the free diacid via hydrolysis of the pNB ester, with no detectable epimerization.
