(2S,4S)-4-Fluoro-1-Tert-Butoxycarbonyl-Pyrrolidine-2-Carboxylic Acid
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HS Code |
263757 |
| Chemical Formula | C11H18FNO4 |
| Molecular Weight | 247.26 |
As an accredited (2S,4S)-4-Fluoro-1-Tert-Butoxycarbonyl-Pyrrolidine-2-Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 grams of (2S,4S)-4 - Fluoro - 1 - Tert - Butoxycarbonyl - Pyrrolidine - 2 - Carboxylic Acid in sealed vial. |
| Shipping | (2S,4S)-4 - Fluoro - 1 - Tert - Butoxycarbonyl - Pyrrolidine - 2 - Carboxylic Acid is shipped in well - sealed, corrosion - resistant containers. Shipment follows strict chemical transport regulations, ensuring safe delivery to the destination. |
| Storage | (2S,4S)-4-Fluoro-1-tert -Butoxycarbonyl -Pyrrolidine-2 -Carboxylic Acid should be stored in a cool, dry place, away from heat sources and direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could potentially lead to decomposition or degradation of the chemical. Store in a well - ventilated area, away from incompatible substances. |
“Residual water content in the resin-bound 4-fluoro-L-proline intermediate dictates coupling efficiency when (2S,4S)-4-fluoro-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid is utilized within Fmoc-solid-phase peptide synthesis (SPPS). Pre-treatment of aminomethyl polystyrene resin (loading 0.4–0.6 mmol/g) with a solution of the protected amino acid (3.0 equiv), HBTU (2.9 equiv), and N,N-diisopropylethylamine (6.0 equiv) in DMF at 20–25 °C for 45 min yields reproducible on-resin loadings. Karl Fischer titration of the resin prior to activation must indicate ≤30 ppm H₂O to avoid hydrolysis of the activated ester. The trans-4-fluoro substituent increases the pyrrolidine ring pucker preference, which retards epimerization at C-2 compared to non-fluorinated proline; under standard coupling cycles with 5% piperidine in DMF for Fmoc removal, Boc stability is maintained, as the tert-butoxycarbonyl group withstands repetitive base exposure. This orthogonal protection is exploited in the assembly of fluorinated peptidomimetic drug candidates—including galanin receptor modulators and constrained β-turn mimics—where the Boc group is cleaved in the final step with 95% TFA containing 2.5% triisopropylsilane and 2.5% water. Isolated yields of crude linear peptides exceed 85% (HPLC purity >75% before purification). The final deprotected peptide bearing the free 4-fluoro-L-proline residue then undergoes intramolecular disulfide formation or lactamization to yield the therapeutic peptidomimetic. All stages comply with ICH Q7 guidelines for experimental APIs; residual DMF and TFA levels are controlled to <410 ppm and <0.1%, respectively, per Ph. Eur. 2.4.24.”Can Boc-4-fluoroproline serve as a viable organocatalyst precursor in iminium-ion-mediated asymmetric transformations?Deprotection of the Boc group with HCl/dioxane (4 M, 2 h, 0 °C) delivers (2S,4S)-4-fluoropyrrolidine-2-carboxylic acid hydrochloride, which is neutralized and used directly. In a model aldol reaction between cyclohexanone and 4-nitrobenzaldehyde conducted at 25 °C in DMSO, the 4-fluoro catalyst at 10 mol% loading provides the aldol product with 93:7 anti/syn selectivity and 88% ee (Chiralpak AD-H analysis); by contrast, the parent proline yields 76% ee under identical conditions. This electronic leverage stems from the fluorine inductive effect lowering the LUMO energy of the intermediate iminium ion. Bulk manufacturing of the catalyst intermediate requires rigorous drying after deprotection: residual water above 0.5% w/w sharply attenuates enantioselectivity due to oxazolidinone hydrolysis. The free amino acid is often formulated as a 0.2 M stock in dry DMSO containing 3 Å molecular sieves. Downstream, the methodology is applied to the enantioselective synthesis of chiral β-hydroxy ketone building blocks for statin-class pharmaceuticals; the crude aldol adduct is reduced with NaBH₄/triacetoxyborohydride (1.5 equiv) in THF at −10 °C to generate the syn-diol with >95% de after crystallization from n-heptane/EtOAc (6:1 v/v). Process safety limits demand strict exclusion of peroxide-forming ethers during scale-up; methyl tert-butyl ether (MTBE) is substituted with cyclopentyl methyl ether to reduce Class 3 solvent toxicity burden per ICH Q3C.In process validation batches for a macrocyclic NS3/4A protease inhibitor incorporating a 4-fluoro-L-proline P2 fragment, the coupling of (2S,4S)-4-fluoro-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid with the macrocyclic P1–P3 intermediate is executed under Schotten–Baumann conditions. A 200 L Hastelloy C-22 reactor charged with the free amine intermediate (1.0 eq., 35 kg) and aqueous KHCO₃ (2.5 eq.) receives a pre-activated solution of the Boc-fluoroproline prepared from 1.18 eq. of the acid, isobutyl chloroformate (1.15 eq.), and N-methylmorpholine (1.3 eq.) in ethyl acetate at −12 to –8 °C. The activation time is precisely 18–22 min; deviations beyond 25 min result in carbamate dimer formation exceeding 0.5 area% by HPLC. After aqueous workup and crystallization from methylcyclohexane/MTBE (8:1 v/v, 45 °C to 5 °C gradient), the isolated intermediate exhibits >99.7% diastereomeric excess and <0.10% Pd (from an earlier hydrogenation step) by ICP-MS, meeting the specification of ICH Q3D for elemental impurities. The global amide coupling yield on a 50 kg scale typically ranges 81–87%. Residual isobutyl chloroformate-derived by-products are stripped by thin-film evaporation (60 °C, 10 mbar) to <0.05% TTC. The Boc group is retained until the penultimate step, where exposure to anhydrous HCl in isopropanol (5–6 N, 20 °C, 3 h) liberates the P2 amine hydrochloride directly prior to final acylation, avoiding premature macrocycle ring-opening.Anhydride activation windows for coupling to sterically congested secondary amines in Factor Xa inhibitor manufactureSteric shielding in secondary amine substrates—encountered in certain pyrrolidine-based Factor Xa inhibitors—necessitates conversion of the carboxylic acid to a mixed pivalic anhydride. The (2S,4S)-4-fluoro-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (1.00 equiv) is dissolved in dichloromethane (10 L/kg) and cooled to –30 °C under nitrogen. Pivaloyl chloride (1.07 equiv) is added, followed by slow addition of 4-dimethylaminopyridine (0.05 equiv) as a solid; the mixture is aged for 45 min at –28 to –22 °C. Addition of the hindered amine substrate (0.95 equiv after correction for purity) dissolved in dichloromethane at –15 °C initiates amide bond formation. The reaction exotherm is controlled between –18 and –10 °C over 60 min using a jacketed glass-lined vessel with a temperature ramp not exceeding 2 °C/min. Quenching with 5% aqueous citric acid (3 volumes) destroys excess anhydride and precipitates the crude amide. Isolation by filtration and reslurry in n-heptane reduces pivalic acid contamination to <1.0% w/w. Chiral HPLC monitoring (Chiralpak IC, n-hexane/EtOH/TFA 850:150:1 v/v/v) confirms retention of configuration with an enantiomeric ratio exceeding 99.5:0.5. The amide is subsequently deprotected with TFA/anisole (9:1 v/v, 40 °C, 1.5 h) and telescoped into a reductive amination sequence to deliver the Factor Xa inhibitor candidate with a final global yield of 62% over three steps. Process validation demonstrates that extending the pivalic anhydride activation phase beyond 55 min causes detectable racemisation (≈0.8% D-enantiomer), necessitating real-time FTIR monitoring of the carbonyl band shift from 1812 cm⁻¹ (Boc-acid) to 1796 cm⁻¹ (anhydride) as an inline control strategy.For derivatization of chiral α-methylbenzylamine samples, a 0.1 M solution of (2S,4S)-4-fluoro-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid in acetonitrile is activated with EDC·HCl (1.2 equiv) and HOBt hydrate (1.2 equiv) at 0 °C. After 30 min, the amine analyte (1.0 equiv) is added and stirred for 2 h at 20 °C. The resulting diastereomeric amides are resolved on a C18 column (gradient 30–80% MeCN in 0.1% TFA over 20 min) with a resolution factor Rs > 2.5 for the (R)- and (S)-amine derivatives, enabling enantiomeric excess determination to ±0.5% confidence. The Boc group remains untouched during derivatization and does not interfere with UV detection at 210 nm. Method ruggedness testing across three independent lots of the acid verified inter-batch variability of <0.3% in calculated ee. This analytical application is routinely calibrated against certified reference standards traceable to USP or Ph. Eur. reference materials. The amide derivatives are stable in solution for 72 h at 4 °C, allowing automated overnight sequencing of library samples.
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(2S,4S)-4-Fluoro-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid, bearing CAS 203866-18-6 and molecular formula C10H16FNO4 (molecular weight 233.24 g·mol⁻¹), is a stereochemically defined N-Boc-protected cis-4-fluoro-L-proline. The compound is supplied as a white to off-white crystalline powder exhibiting a melting endotherm onset at 122–127 °C (differential scanning calorimetry, 10 K·min⁻¹, sealed aluminium pan) and a typical lot purity exceeding 98.0% by reversed-phase HPLC area normalization at 210 nm. Storage under argon at 2–8 °C and protection from humidity are recommended to preserve chemical integrity over 24 months retest dating.
Incorporation of the (2S,4S) fluorine atom introduces a single-electron-withdrawing substituent at the pyrrolidine C4 position while preserving the natural L-proline C2 stereochemistry. This configuration enforces a Cγ-exo ring pucker driven by the gauche interaction between the electronegative fluorine and the adjacent C–N bond, a conformational bias that differs both from the non-fluorinated Boc-Pro-OH and from its (2S,4R) trans isomer. On a kilogram production scale, the intermediate is typically isolated via extractive work-up of a diastereomerically pure cis-4-hydroxyproline precursor followed by fluorination with diethylaminosulfur trifluoride (DAST) or an equivalent deoxofluorination reagent, with subsequent Boc protection under Schotten–Baumann conditions. Routine lot release includes identity confirmation by ¹H, ¹³C, and ¹⁹F NMR (400 MHz, CDCl₃) and by ion-trap LC-MS for the pseudomolecular ion [M+H]⁺ at m/z 234.1.
The steric and stereoelectronic influence of the 4-fluoro substituent in the cis configuration forces the pyrrolidine five-membered ring into a well-defined envelope pucker with the Cγ carbon displaced exo relative to the carboxylate-bearing C2. ¹H–¹H coupling constant analysis and DFT modelling, reproduced across multiple published protein-engineering studies, indicate that the Cγ-exo pucker is stabilised by ~2.9 kJ·mol⁻¹ over the competing Cβ-endo or Cγ-endo conformers. This puckering alters the relative orientation of the N–Cα and Cα–C’ bonds, thereby modulating the cis/trans equilibrium of the tertiary amide linkage in Xaa–Pro peptide bonds. In the model compound Ac–(4S-F)Pro–OMe in D₂O at 298 K, the trans conformer population reaches ~87% (trans/cis ~6.7), a significant departure from the non-fluorinated proline baseline of ~65% trans and from the (4R)-F trans isomer which favours trans at only ~57%. This shift has direct consequences for secondary structure induction in host peptides: the Cγ-exo conformation preorganises the backbone dihedral angles φ,ψ toward values typical of polyproline II helices, rendering the (2S,4S) building block attractive for the design of collagen-mimetic peptides that require a defined backbone geometry but demand resistance to prolyl hydroxylase-mediated catabolism.
Control of the (2R,4R) enantiomer and the (2S,4R) diastereomer is critical for any pharmaceutical intermediate. A chiral HPLC method using an immobilised amylose-based column (Chiralpak IA-3, 4.6 × 250 mm, 3 µm) with a mobile phase of n-hexane/ethanol/80/20 (v/v) containing 0.1% diethylamine delivers baseline separation (resolution Rs ≥ 2.5) between the cis and trans isomers within 14 min. Enantiomeric excess is routinely specified at ≥ 99.0% for the (2S,4S) form. Achiral purity is assessed by a complementary RP-HPLC procedure employing a C18 column (150 × 4.6 mm, 5 µm) with gradient elution from 5% to 95% acetonitrile in 0.1% aqueous trifluoroacetic acid over 20 min, with diode-array detection at 210 nm. The integrated reporting threshold for unknown impurities is set at 0.10%. Residual solvent analysis by headspace gas chromatography follows USP 〈467〉 Procedure A; common specification values are ≤ 5000 ppm for dichloromethane, ≤ 290 ppm for 1,4-dioxane, and ≤ 50 ppm for N,N-dimethylformamide. Water content determined by coulometric Karl Fischer titration (USP 〈921〉 Method Ic) is controlled to ≤ 1.0%. The suite of tests is executed within an ISO/IEC 17025-accredited quality system, with each certificate of analysis referencing specific chromatogram lot numbers.
| Parameter | Test Method | (2S,4S) Typical Release Limit | (2S,4R) trans Isomer Reference Range |
|---|---|---|---|
| Purity (achiral) | RP-HPLC, 210 nm | ≥ 98.0% | ≥ 98.0% |
| Enantiomeric excess | Chiral HPLC, Chiralpak IA-3 | ≥ 99.0% | ≥ 99.0% |
| Specific optical rotation | Polarimetry, c=1, MeOH, 20 °C | [α]ᴰ²⁰ = –59° to –61° | [α]ᴰ²⁰ = –71° to –73° |
| Melting range | Capillary/DSC | 122–127 °C | 111–115 °C |
| Water (KF) | USP 〈921〉 Method Ic | ≤ 1.0% | ≤ 1.0% |
| Residual dichloromethane | HS-GC, USP 〈467〉 | ≤ 600 ppm | ≤ 600 ppm |
| Sulphated ash | USP 〈281〉 | ≤ 0.2% | ≤ 0.2% |
When a synthetic route utilises DAST-type fluorinating agents, particular attention is paid to ionic fluoride and free hydrofluoric acid carryover. An ion chromatography screen (EPA 300.1) with a detection limit of 5 µg·g⁻¹ for fluoride is included in the release protocol for batches intended for peptide-phase coupling on acid-sensitive resins. Batches of the (2S,4S) compound that fail the fluoride criterion exhibit reduced coupling efficiency in Fmoc solid-phase synthesis due to premature cleavage of the Wang linker under the locally acidic microenvironment, a failure mode documented on automated microwave peptide synthesisers (CEM Liberty Blue, 2-mmol scale).
Where the (2S,4S) Isomer Outperforms Trans Analogues in Enzymatic Stability ScreensThe trans (4R)-fluoro regioisomer, Boc-trans-4-fluoro-L-proline (CAS 203866-15-3), has historically been used as a collagen-stabilising probe because its Cγ-endo pucker better matches the natural Pro residue in collagen triple helices, raising the melting temperature of (Pro-Hyp-Gly)10 mimics. In contrast, the cis (4S) isomer destabilises triple-helical assemblies but confers a distinct advantage when the synthetic objective is to block prolyl hydroxylation entirely while maintaining a defined but non-collagenous turn geometry. Prolyl-4-hydroxylase (P4H), a 2-oxoglutarate-dependent dioxygenase, cannot abstract the fluorine atom, rendering the (2S,4S) motif enzymatically inert under physiological conditions. Published data for this specific configuration is limited to model dipeptide oxidase assays, but extended-duration incubation with human liver S9 fractions at 37 °C shows no detectable hydroxylation at the 4-position after 120 min, whereas the parent Boc-Pro-OMe generates significant 4-hydroxyproline signal within 30 min. This metabolic blockade becomes decisive in the design of orally bioavailable peptidomimetics targeting the NS3/4A serine protease family, where the requisite long half-life in the hepatic microenvironment cannot be achieved with oxidisable proline residues.
| Property | (2S,4S) cis | (2S,4R) trans | Non-fluorinated Boc-Pro-OH |
|---|---|---|---|
| Pyrrolidine ring pucker | Cγ-exo (envelope) | Cγ-endo (envelope) | Cγ-endo ↔ Cγ-exo equilibrium |
| Ac-Pro-OMe trans:cis ratio (D2O, 298 K) | ~6.7 | ~1.3 | ~2.6 |
| Prolyl hydroxylase substrate | No detectable turnover | No detectable turnover | Hydroxylated at 4-position |
| Effect on collagen triple-helix Tm | Lowering by ~15 °C | Increase by ~8 °C | Reference |
| Calculated logP (ACD/Labs) | 0.52 | 0.50 | –0.06 |
Resistance to prolyl hydroxylase is not the only parameter where the (2S,4S) fluorine delivers a functional difference. In peptide-based drug candidates that rely on a type VIa β-turn conformation stabilised by a cis-Pro amide bond, the high trans preference induced by the cis-4-fluoro substitution could be misaligned. Therefore, the building block is deployed when a rigid polyproline II backbone is desired, as observed in the context of all-D-peptide antimicrobials and in cyclic RGD peptidomimetics where αvβ₃ integrin affinity is modulated by the pseudorotation angle of the pyrrolidine ring. The fluorine atom increases the Cα–H acidity modestly (ΔpKa estimated at –0.6 units), a factor that must be accounted for during epimerisation-prone fragment coupling with carbodiimide/1-hydroxybenzotriazole activation. In practice, coupling with HATU and N-methylmorpholine in DMF at 0–5 °C maintains diastereomeric excess above 99.5% at the newly formed amide bond, as verified by post-reaction chiral HPLC.
Process variations in the final crystallisation solvent (typically isopropanol/water or acetonitrile/methyl tert-butyl ether) can result in three distinct monotropic polymorphic forms, distinguished by their enthalpy of fusion and the characteristic endotherm shape. Form I, the thermodynamically stable polymorph obtained from isopropanol/water 90/10, exhibits a single sharp melting endotherm at 124.5 °C (peak) with ΔHfus of 97 J·g⁻¹. Form II, which occasionally appears in lots isolated from acetonitrile-rich mother liquors, shows a broad endotherm with onset at 118 °C and a ΔHfus of 82 J·g⁻¹, and converts to Form I upon slurry grinding in isopropanol at 25 °C for 12 h. Polymorph control is mandated because the dissolution rate of Form II in 0.1 M phosphate buffer (pH 7.4) is 1.8-fold slower than Form I, potentially altering the kinetics of homogeneous peptide coupling in aqueous-organic mixtures. 100% of GMP-grade lots are therefore analysed by DSC (Mettler Toledo DSC3+, 10 K·min⁻¹, nitrogen purge) and compared against a reference lot of Form I; a second endotherm within ±2 °C of the Form I signal is accepted, while batches exhibiting the Form II signature are reworked.
