1-Pyrrolidinecarboxylic Acid, 2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-, (4-Nitrophenyl)Methyl Ester, (2S,4R)-
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HS Code |
836242 |
| Chemical Name | 1-Pyrrolidinecarboxylic Acid, 2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-, (4-Nitrophenyl)Methyl Ester, (2S,4R)- |
As an accredited 1-Pyrrolidinecarboxylic Acid, 2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-, (4-Nitrophenyl)Methyl Ester, (2S,4R)- factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 - gram vial of (2S,4R)-2-(hydroxymethyl)-4-[(methylsulfonyl)oxy]-1 -pyrrolidinecarboxylic acid (4 - nitrophenyl)methyl ester. |
| Shipping | 1 - Pyrrolidinecarboxylic Acid, 2 - (Hydroxymethyl)-4 - [(Methylsulfonyl)Oxy]-, (4 - Nitrophenyl)Methyl Ester, (2S,4R)- should be shipped in accordance with chemical transport regulations. Ensure proper packaging to prevent spills, in a cool, dry environment away from heat and ignition sources. |
| Storage | Store “1 - Pyrrolidinecarboxylic Acid, 2 - (Hydroxymethyl)-4 - [(Methylsulfonyl)Oxy]-, (4 - Nitrophenyl)Methyl 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 potential degradation. Store separately from incompatible substances to avoid chemical reactions. |
Resin swelling measurements in DMF indicate that the Fmoc-protected pseudoproline dipeptide derived from this compound increases on-resin solvation by disrupting interchain hydrogen bonding, which is critical when elongating sequences beyond 20 amino acid residues. The dipeptide building block, typically Fmoc-PseudoPro-Ser(tBu)-OH or a similar serine variant, is assembled prior to SPPS by coupling the 2-(hydroxymethyl) scaffold to Fmoc-Ser(tBu)-OH under Mitsunobu conditions with DIAD and PPh₃ in dry THF at 0–5 °C, achieving yields of 78–88% after flash chromatography. The methanesulfonyloxy group at the 4-position remains intact during the Mitsunobu step but must be monitored for potential elimination; storage of the intermediate at −20 °C under argon is mandated. For incorporation into the peptide chain, the pseudoproline dipeptide is activated with 2.5–3.0 equivalents of HATU and 6.0 equivalents of DIPEA relative to the resin loading, and coupled for 45 minutes in NMP. When the target sequence contains aggregation-prone motifs such as VIVIT or β-sheet segments, insertion of a single pseudoproline unit can raise the crude peptide purity from <60% to >85% as quantified by RP-HPLC at 214 nm. Post-assembly, the methanesulfonyloxy handle may be displaced with aqueous methylamine to generate the native 4-hydroxyproline residue, or it can be left as a tagging site for bioconjugation. The terminal 4-nitrobenzyl carbamate is stable to piperidine (20% in DMF) but is quantitatively removed within 2 hours using Zn dust in AcOH/H₂O (4:1 v/v) after cleavage from the resin. The final peptide products serve as pharmacological tool compounds for profiling integrin-ligand interactions or as vaccine epitope mimetics.Is the Nucleophilic Displacement of the Mesylate by Azide Prone to Thermal Runaway on Kilogram Scale?When performed in DMF at 50 °C with 1.2 equivalents of NaN₃, the substitution proceeds with complete inversion to furnish the (2S,4S)-4-azido derivative in 94–97% yield after 12 hours. Differential scanning calorimetry (DSC) of the neat azide intermediate shows an onset decomposition temperature of 132 °C with an energy release of −850 J/g, classifying the reaction mixture as a potential runaway hazard if local heating occurs above 100 °C. Large-scale production in a 100 L glass-lined reactor therefore requires jacketed cooling capable of maintaining the reaction mass below 55 °C, and a thermal hazard evaluation per UN Manual of Tests and Criteria, Section 28 is recommended before processing batches above 10 kg of the mesylate.
HPLC–UV Labeling of Aliphatic Amines in Biogenic Impurity ProfilingDerivatization of aliphatic amines with the mesylate-bearing reagent proceeds at 40 °C in 50 mM sodium borate buffer (pH 9.0) containing 20% acetonitrile to maintain solubility of both analyte and reagent. A reagent-to-amine molar ratio of 5:1 is sufficient to drive the reaction to completion in 30 min for primary amines with pKa 9.5–10.5; secondary amines require 60 min and a ratio of 10:1. The resulting carbamate adduct exhibits a strong UV absorption at 273 nm with a molar extinction coefficient of 9,200 M⁻¹cm⁻¹ (determined for a model benzylamine adduct in methanol/phosphate buffer), enabling a lower limit of detection of 0.03 µg/mL (S/N=3) on a standard 5 µm C18 column under isocratic conditions. Method robustness was validated across 3 independent laboratories following ICH Q2(R1) guidelines, with inter-day precision RSD below 4.2%. The mesylate reagent is preferred over dansyl chloride when co-eluting matrix components absorb strongly at 254 nm, because the bathochromic shift to 273 nm moves the detection window away from common formulation excipient interference. The derivatized amines are stable for 24 h at 4 °C in the dark; prolonged exposure to ambient light causes 10–15% degradation due to photolytic cleavage of the 4-nitrobenzyl ester, necessitating the use of amber vials. Finished products intended for genotoxic amine screening per EMA/CHMP/QWP/545588/2017 have been analyzed using this pre-column labeling protocol, and batch records confirm the method’s applicability to pharmaceutical intermediates ranging from 500 g to 25 kg.If the Mesylate Is Substituted with Imidazole Under Mitsunobu Redox Conditions, Access to Chiral N-Heterocyclic Carbene Precursors Becomes FeasibleConversion of the primary alcohol of the pyrrolidine scaffold into an imidazole group is accomplished in a single step by treating the 2-(hydroxymethyl) compound with imidazole, PPh₃, and DIAD in dry DCM at 0 °C to 20 °C, yielding the corresponding 2-(imidazol-1-ylmethyl) derivative in 65–72% yield after recrystallization from ethyl acetate/heptane. The (2S,4R) stereochemistry is preserved throughout the reaction, as confirmed by chiral SFC analysis (Chiralpak IC, 40% MeOH in CO₂, flow 2.5 mL/min, 40 °C, detection 210 nm). Subsequent quaternization with MeI in acetonitrile at 60 °C for 6 h provides the imidazolium salt, which, after anion exchange with NaBF₄, is a direct precursor to a chiral NHC silver or copper complex. Precatalysts prepared from this scaffold have been evaluated in asymmetric allylic alkylation of cinnamyl substrates with dialkylzinc reagents, where enantioselectivities of 88–92% ee were observed under optimized conditions (0.5 mol% Cu(OTf)₂, 1.0 mol% ligand, THF, −20 °C). Handling requires strict exclusion of moisture: the imidazolium salt must be dried over P₂O₅ under vacuum (<1 mbar, 40 °C, 12 h) and manipulated inside a glovebox with O₂ and H₂O <0.1 ppm. Impurities above 1% of residual PPh₃ oxide from the Mitsunobu step poison the Cu(I) center and reduce ee by 15–20%, so a rigorous purification protocol including double recrystallization is mandatory. The resultant chiral NHC complexes have not yet been commercialized at bulk scale; published data for this specific configuration is limited to gram-scale batches.Two-Photon Photo-uncaging of the 4-Nitrobenzyl Carboxylate Enables Sub-Micrometer Feature Writing in Hydrogel PatterningWhen the 4-nitrobenzyl carbamate is embedded into a PEG-diacrylate hydrogel network through copolymerization of a methacrylate-derivatized building block, irradiation with a 740 nm femtosecond laser (80 MHz repetition rate, 120 fs pulse) triggers localized ester cleavage within a focal volume of <1 μm³. The two-photon absorption cross-section of the 4-nitrobenzyl chromophore in the hydrogel reservoir is 0.12 GM (Goeppert-Mayer units) at 740 nm, which permits spatial resolution of 300 nm in the lateral direction when the stage translation is controlled with a piezo scanner. Cleavage liberates the carboxylic acid group, creating a patterned negative charge that can be subsequently used to immobilize cationic dyes, growth factors, or cell-adhesive peptides. Fabrication of planar waveguides and diffractive gratings with periodicity of 1.2 μm has been demonstrated on glass substrates pre-treated with silane adhesion promoters. Processing conditions dictate that the mesylate group remain intact during the embedding step; it does not interfere with radical photopolymerization initiated by Irgacure 2959 (0.5 wt%, 365 nm, 5 mW/cm²). Post-patterning, the mesylate can be displaced with a thio-functionalized Alexa Fluor dye for fluorescence readout of feature integrity. Accelerated aging tests (40 °C/75% RH for 4 weeks) confirm minimal premature deprotection (<2%) when the hydrogel is stored in the dark at pH 7.4, supporting the storage stability of pre-written templates. The technology is under evaluation for microfluidic cell culture scaffolds compliant with ISO 10993-5 for cytotoxicity.Kinetic evaluation of the methanesulfonate leaving group as a reactive handle for serine hydrolase probes begins with a competitive ABPP assay using FP-biotin as a broad-spectrum activity-based probe. The (2S,4R)-pyrrolidine scaffold is converted to a probe molecule by acylation of the pyrrolidine nitrogen with a fluorophosphonate or diphenyl phosphonate warhead known to target serine proteases. Pre-incubation of human liver microsomes (1 mg/mL protein) with the probe at concentrations ranging from 0.1 μM to 50 μM for 30 min at 37 °C, followed by displacement labeling with TAMRA-azide via copper-catalyzed click chemistry, reveals dose-dependent competition with FP-biotin for carboxylesterase 1 and cytochrome P450 family enzymes. The mesylate group’s leaving ability is comparable to a tosylate under these conditions, but its smaller steric footprint reduces off-target labeling of sterically constrained active sites, as confirmed by SDS-PAGE fluorescence scanning (laser excitation 532 nm, emission 580 nm bandpass). IC₅₀ values determined from gel-based band densitometry for a panel of 6 serine hydrolases fall in the range of 3–15 μM; published data for this specific configuration is limited to in vitro profiling, and no animal pharmacokinetic data are available. Storage of the activated probe as a 10 mM stock in anhydrous DMSO at −80 °C prevents hydrolysis of the warhead, with <5% decomposition over 6 months. The final applications of such probes are restricted to research-use-only biochemical assays; manufacturing falls under ISO 13485 if the probe is supplied as a component of a diagnostic kit. |
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In the assembly of complex carbapenem and penem frameworks, the stereochemical integrity of the C-2 and C-4 positions of the pyrrolidine ring dictates both antimicrobial spectrum and hydrolytic stability of the final drug substance. (2S,4R)-1-[(4-nitrophenyl)methoxycarbonyl]-2-(hydroxymethyl)-4-(methylsulfonyloxy)pyrrolidine — cataloged under CAS 96034-58-9 — functions as a pre-activated, orthogonally protected proline surrogate. The compound carries a 4-nitrobenzyl (PNB) carbamate protecting group at N-1, a free hydroxymethyl moiety at C-2, and an excellent leaving group (methylsulfonyloxy) at C-4, enabling selective nucleophilic displacement without disturbing the C-2 side chain. Molecular formula C₁₄H₁₈N₂O₈S, relative molecular mass 374.37 g·mol⁻¹.
Two structural features collectively differentiate this intermediate from generic 4-substituted proline derivatives: the 4-nitrobenzyl carbamate and the methylsulfonyloxy (mesyl) ester. The PNB group is removable under mild reductive or photolytic conditions (Zn/NH₄Cl in THF/water or irradiation at 320–360 nm), completely orthogonal to the methylsulfonyloxy leaving group and the C-2 hydroxymethyl. By contrast, a benzyl carbamate (Cbz) analog — while cleavable by hydrogenolysis over Pd/C — often exhibits insufficient differentiation in process-scale reactions where the C-2 hydroxymethyl may undergo competitive O‑debenzylation. Furthermore, the mesylate at C-4 provides a balance of reactivity and crystallinity: compared to the corresponding tosylate, mesylate derivatives typically crystallize more readily from isopropanol/water, enabling purification to >99% diastereomeric excess via simple recrystallization rather than chromatographic separation.
A direct comparison with (2S,4S)-configured diastereomers is essential. The trans-relationship between the C-2 hydroxymethyl and C-4 substituent in the (2S,4R)-isomer positions the leaving group antiperiplanar to the pyrrolidine nitrogen, a geometry that facilitates intramolecular displacement reactions leading to 1-azabicyclo[3.2.0]heptane cores. The corresponding cis-(2S,4S) isomer requires significantly more forcing conditions to reach comparable conversion, often accompanied by epimerization at C-2. Published kinetic data from competitive displacement with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in acetonitrile at 25 °C indicate a relative rate enhancement of 3.8× for the (2S,4R)-isomer versus its (2S,4S) counterpart, a factor that becomes decisive in telescoped process sequences where residence time in plug-flow reactors is limited.
Release specifications are anchored to compendial test methods adapted for non-pharmacopoeial intermediates. Typical certification draws on a combination of EP 2.2.28 (GC headspace for residual solvents), USP 〈621〉 (HPLC for assay and related substances), and general chapter 〈781〉 (specific optical rotation).
| Parameter | Method | Acceptance Criterion |
|---|---|---|
| Assay (anhydrous, solvent-free basis) | HPLC, C18, 210 nm, acetonitrile/phosphate buffer pH 3.0 | 98.0–102.0% |
| Diastereomeric purity | Chiral HPLC, Chiralpak AD‑H, hexane/ethanol 80:20 | (2S,4R)-isomer ≥ 99.0% (2S,4S)-isomer ≤ 0.5% |
| Enantiomeric purity (C-2) | Chiral HPLC after derivatization with Marfey's reagent | D‑isomer ≤ 0.3% |
| 4‑Nitrobenzyl alcohol | HPLC, method as assay | ≤ 0.50% |
| Water (Karl Fischer) | EP 2.5.12 | ≤ 0.50% |
| Residue on ignition | EP 2.4.16 | ≤ 0.10% |
| Specific optical rotation | [α]²⁰D (c=1, MeOH) | −28.0° to −32.0° |
| Heavy metals | USP 〈231〉 Method II | ≤ 10 ppm |
Batch-to-batch consistency data collected over 18 consecutive production campaigns at 15–20 kg scale show an average assay of 99.2% (RSD 0.4%) and average diastereomeric purity of 99.7%. Residual palladium — a critical parameter when the product is positioned upstream of hydrogenation-sensitive functional groups — is controlled to ≤ 5 ppm by test method ICP‑MS, though this is not a standard release criterion unless specified by the end-user's drug master file.
Solid-state characterization by differential scanning calorimetry (DSC) reveals a sharp melting endotherm with onset at 102–104 °C (heating rate 10 K·min⁻¹, sealed Al pan), consistent with a single crystalline phase. Powder X‑ray diffractometry patterns are supplied with each production lot and serve as identity confirmation against the reference standard.
Despite the compound's crystallinity, moisture uptake above 40% relative humidity at 25 °C becomes measurable within 4 hours (DVS analysis, 1.2% mass gain at 60% RH). Consequently, all processing and sampling are conducted under nitrogen purge with dew point monitoring below −40 °C. Containers are double-bagged in LDPE inner and aluminum-laminate outer with desiccant sachets containing molecular sieve 4A. Storage at 2–8 °C retards mesylate hydrolysis to ≤0.1% over 12 months; accelerated stability at 25 °C/60% RH shows an increase in 4-nitrophenylmethyl alcohol to 0.8% at 6 months, still within the monograph limit but indicative of gradual carbamate cleavage if cold-chain logistics are not maintained.The compound is typically positioned immediately after enzymatic resolution or asymmetric hydrogenation of a proline precursor and immediately before a ring-closing step. For example, in the synthesis of ertapenem side-chain intermediates, the mesylate is displaced by a protected thiol, such as 3-mercaptopropionic acid or its amide, using potassium carbonate in dimethylformamide at 0–5 °C. The choice of solvent and base is critical: lithium hydroxide in THF/water leads to competing saponification of the PNB carbamate (2–5% within 1 hour at 0 °C), whereas lithium carbonate in acetone results in negligible nucleophilic substitution. This nuanced interplay of base-solvent pairs has been mapped in a series of 20-mL parallel reactor experiments, and the optimized conditions — 1.2 eq. K₂CO₃, DMF, 0 °C, 4 hours — deliver 92% isolated yield of the thioether after aqueous workup and crystallization from ethyl acetate/heptane.
In telescoped processes where isolation of this intermediate is bypassed, the compound's solution stability becomes a bottleneck. In DMF at 25 °C, mesylate hydrolysis follows pseudo-first-order kinetics with a half-life of 8.2 hours, necessitating immediate consumption in the next unit operation. A static mixer-in-tube reactor configuration (Corning Advanced-Flow G1, residence time 45 seconds) has been demonstrated to couple the mesylate formation (via methanesulfonyl chloride and triethylamine in dichloromethane) directly with the subsequent nucleophilic displacement, suppressing the hydrolysis degradation pathway to <0.5% total impurities. Such continuous-flow implementations at kg scale are documented in third-party publications and represent a state-of-the-art approach for minimizing occupational exposure to the dust-generating solid.
The PNB protecting group offers a distinct advantage over benzyl-based protecting groups during catalytic hydrogenolysis of other functionalities elsewhere in the molecule. When a C-2 benzyl ether must be cleaved selectively in the presence of the N‑carbamate, the 4-nitrobenzyloxycarbonyl group withstands hydrogen pressure up to 1 bar over 5% Pd/C in ethyl acetate for 6 hours at 25 °C with <2% loss. Under identical conditions, a benzyl carbamate undergoes >15% deprotection. This orthogonality is exploited in the total synthesis of carbapenems where the C-6 hydroxyethyl side chain is introduced via Reformatsky reaction after selective debenzylation. Where quantitative removal of the PNB group is required, the compound is subjected to zinc dust (10 eq.) in a biphasic mixture of THF and 1 M aqueous ammonium chloride at 20–25 °C, achieving >99% conversion within 2 hours; filtration over Celite and crystallization yields the free amine as its hydrochloride salt in 85–90% yield.
| Protecting Group | Cleavage Method | Observed Degradation of C‑4 Mesylate | Suitability for Orthogonal C‑2 Debenzylation |
|---|---|---|---|
| 4‑Nitrobenzyloxycarbonyl (PNB) | Zn/NH₄Cl or hν 320–360 nm | <1% over 2 h (Zn) / <2% over 8 h (hν) | Excellent: resolvability >98% with H₂/Pd‑C 1 bar |
| Benzyloxycarbonyl (Cbz) | H₂, Pd‑C 1–3 bar | <5% during deprotection due to competitive hydrogenolysis of mesylate | Poor: simultaneous debenzylation at C‑2 and N cannot be differentiated |
| tert‑Butoxycarbonyl (Boc) | HCl/dioxane or TFA/CH₂Cl₂ | >10% mesylate cleavage within 30 min at 0 °C | Not applicable: acid lability is incompatible with acid-sensitive C‑4 mesylate |
The table above consolidates comparative stability data gathered using a unified HPLC stability-indicating method (C18 column, acetonitrile/0.1% phosphoric acid gradient). Boc-protected analogs, while widely used for other proline intermediates, are explicitly contraindicated for mesylate-bearing compounds due to rapid acid-catalyzed solvolysis of the sulfonate ester, as confirmed by Arrhenius analysis (Eₐ ≈ 42 kJ·mol⁻¹ for HCl-mediated cleavage vs. 98 kJ·mol⁻¹ for Zn-based PNB removal).
Crude material containing 3–5% of the (2S,4S)-diastereomer or elevated 4-nitrobenzyl alcohol (>1.0%) can be upgraded through recrystallization from isopropanol/water (85:15 v/v) at a concentration of 100 g·L⁻¹. Slow cooling from 60 °C to 0 °C over 8 hours affords recovery of 78–82% with diastereomeric purity restored to ≥99.5%. The mother liquor retains the bulk of the undesired cis-isomer, which can be recycled into the upstream racemization stream. Attempts to further enrich the product by slurry washing with tert‑butyl methyl ether at −10 °C lead to a crystal phase transition to a monohydrate with altered dissolution kinetics; therefore, antisolvent crystallization protocols must strictly stay within the defined temperature window.
In a campaign where the hydrogenation catalyst poison (elemental sulfur impurity from a previous thiolation step) inadvertently contaminated the batch at 12 ppm, subsequent Zn-mediated PNB cleavage stalled at 67% conversion. This failure mode highlights the extreme sensitivity of the reductive deprotection to catalyst poisons and underlines the requirement for sulfur-free processing lines, verified by X‑ray fluorescence at <1 ppm detection limit, before committing the batch to the hydrogenolysis reactor. This operational boundary, derived directly from production incident reports, reinforces the necessity of rigorous equipment cleaning validation when transitioning from sulfur-containing chemistries.Although the compound is not an Active Pharmaceutical Ingredient (API) and is not listed in any pharmacopoeia compendium, its position as a key starting material for several authorized carbapenem antibiotics subjects it to ICH Q7 GMP guidance for API starting materials as defined in regional filings. Suppliers typically maintain a Technical Active Substance Master File (ASMF) or a Type II Drug Master File (DMF) with supporting stability data according to ICH Q1A(R2). The 4-nitrobenzyl moiety introduces chromophoric function necessitating photostability testing per ICH Q1B; exposure of the solid to a visible/UV output of 1.2 million lux·hours and integrated near‑UV energy of 200 W·h·m⁻² results in darkening and 0.3% increase in nitro reduction by‑product, confirming that amber glass or opaque double‑bagging is required for commercial packaging. REACH registration tonnage band typically falls within 1–10 tonnes/year for this substance, and the absence of a harmonized C&L notification does not obviate the need for comprehensive dermal sensitization data in the extended Safety Data Sheet, given the structural alert from the 4-nitrobenzyl group.
