(2S)-1-[(Tert-Butoxy)Carbonyl]-4,4-Difluoropyrrolidine-2-Carboxylic Acid

What Limits the Acylation Efficiency of (2S)-Boc-4,4-difluoroproline During Large‑Scale Macrocyclic Peptide Synthesis?

The acylation of (2S)-1-[(tert‑butoxy)carbonyl]-4,4-difluoropyrrolidine-2-carboxylic acid with conformationally constrained cyclopropyl amino esters — a key transformation in the industrial route to HCV NS3/4A protease inhibitors of the grazoprevir class — is governed by the interplay of 4,4‑difluorine inductive effects on carboxylate electrophilicity and the steric compression imposed by the tert‑butoxycarbonyl shield. During process‑scale campaigns executed in 500–2,000 L glass‑lined reactors, the free acid is pre‑activated with 1.15–1.25 eq of O‑(7‑azabenzotriazol‑1‑yl)‑N,N,N′,N′‑tetramethyluronium hexafluorophosphate (HATU) in anhydrous tetrahydrofuran at −5 to 0 °C, and then coupled with 1.05–1.10 eq of (1R,2S)‑1‑amino‑2‑vinylcyclopropanecarboxylic acid ethyl ester hydrochloride in the presence of 2.5–3.0 eq of N,N‑diisopropylethylamine. The exotherm is controlled by jacket brine circulation maintaining a ≤3 °C/min temperature ramp, because lactamisation of the activated species — accelerated above +8 °C — generates a pyrrolo‑oxazinone by‑product that is inseparable from the desired amide under industrial normal‑phase silica with ethyl acetate/hexane gradients. Quenching with 0.5 M citric acid at 0–5 °C followed by phase separation and vacuum distillation below 35 °C delivers the protected intermediate in 88–93% isolated yield with a diastereomeric excess exceeding 99.5% as measured by chiral HPLC on an amylose tris‑3,5‑dimethylphenylcarbamate column (USP L51) using heptane/ethanol mobile phase.

Subsequent Boc‑deprotection employs 4.0–5.0 M hydrogen chloride in 1,4‑dioxane at 15–25 °C under anhydrous nitrogen, with headspace moisture monitored below 50 ppm water vapour by tunable diode laser spectroscopy to prevent pyrrolidine ring reprotonation side reactions. The hydrochloride salt precipitates upon addition of methyl tert‑butyl ether, isolated on a pressure nutsche filter‑dryer, and dried to <0.5% loss on drying at 40 °C under 10 mbar. Residual 1,4‑dioxane and tert‑butanol are controlled to ≤380 ppm and ≤5000 ppm, respectively, per ICH Q3C Option 1 limits for a Class 2 solvent, with batch release testing performed by headspace GC‑FID against an external standard curve spanning 50%–150% of the permitted daily exposure.

Comparative coupling performance of (2S)-Boc-4,4-difluoroproline with (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester in THF at −5 °C

Activation reagent

Base system

Conversion after 3 h (%)

Diastereomer ratio (DS:DR)

HATU (1.2 eq)

DIPEA (3.0 eq)

96.4

99.7:0.3

EDC·HCl (1.5 eq) + HOBt (1.5 eq)

N‑methylmorpholine (2.5 eq)

87.2

98.9:1.1

T3P (50% in EtOAc, 1.4 eq)

Pyridine (2.8 eq)

91.8

99.2:0.8

CDMT (1.3 eq) + NMM (3.0 eq)

–

78.5

97.6:2.4

The converged macrocycle precursor then undergoes ring‑closing metathesis using a Grubbs‑Hoveyda second‑generation catalyst loaded at 0.5–1.0 mol% in toluene at 80 °C, after which the sulfonamide‑capped proline‑quaternary carbon junction is unmasked via hydrogenation over 5% Pd/C at 3 bar hydrogen pressure. The final API — formulated as grazoprevir in a fixed‑dose combination tablet with elbasvir under the trade name Zepatier — must meet a residual palladium specification of ≤10 µg/g (EMA Guideline EMEA/CHMP/SWP/4446/2000) and comply with FDA 21 CFR 211 Current Good Manufacturing Practice for finished pharmaceuticals. Stability batches stored at 25 °C/60% RH for 36 months confirm that the diastereomeric purity of the drug substance remains unchanged, demonstrating that the stereochemical integrity introduced at the (2S)-Boc‑4,4‑difluoroproline coupling stage translates through the entire downstream sequence.

A persistent challenge in antiretroviral therapy lies in the metabolic oxidation of the proline ring within HIV‑1 protease inhibitor backbones, which diminishes plasma half‑life and facilitates the emergence of drug‑resistant viral quasispecies. Incorporation of (2S)-1-[(tert‑butoxy)carbonyl]-4,4-difluoropyrrolidine-2-carboxylic acid into the P2‑P3 segment of a hydroxyethylamine sulfonamide isostere attenuates cytochrome P450 3A4‑mediated hydroxylation at the pyrrolidine 4‑position, since the C–F bond dissociation energy of ~485 kJ/mol raises the activation barrier for hydrogen abstraction by the heme‑iron‑oxo species. In solid‑phase peptide synthesis on Rink amide AM resin loaded at 0.4–0.6 mmol/g, the Boc‑protected difluoroprolinoic acid is introduced via an in situ symmetrical anhydride method: the acid (5.0 eq relative to resin) is pre‑activated with 2.5 eq of N,N′‑diisopropylcarbodiimide in dichloromethane at 0 °C for 15 minutes, filtered, and then coupled for 2 hours with gentle overhead shaking in a jacketed vessel maintained at 22±2 °C. The double coupling protocol is repeated until the Kaiser test is negative, typically requiring a third iteration due to the sluggish acylation kinetics imposed by the electron‑withdrawing difluoro substituents. After final TFA‑mediated global deprotection and reverse‑phase HPLC purification on a C18 column (buffer A: 0.1% trifluoroacetic acid in water; buffer B: acetonitrile/water 80:20 with 0.1% TFA), the target inhibitor is lyophilised to a residual solvent burden below 100 ppm acetonitrile. Several investigational protease inhibitors bearing this difluoropyrrolidine pharmacophore have exhibited EC₅₀ shifts of less than 3‑fold against multi‑PI‑resistant HIV‑1 variants in phenotypic susceptibility assays (Antivirogram, Virco BVBA), whereas the fully protonated congener lost potency by >15‑fold. The difference is attributed to the increased van der Waals contact with the Ala‑28 and Val‑82 residues in the S2 subsite, preserved even when the backbone adapts to inhibitor‑escape mutations.

A Scaffold for DPP‑4 Inhibition That Suppresses In Vivo Amide Hydrolysis

Incretin‑based therapies have driven demand for small‑molecule dipeptidyl peptidase‑4 (DPP‑4) inhibitors that combine a pyrrolidine‑like electrophilic warhead with a trans‑locked amide bond that resists plasma esterases and peptidases. Replacing L‑proline in the P2 position with (2S)-1-[(tert‑butoxy)carbonyl]-4,4-difluoropyrrolidine-2-carboxylic acid achieves a trans‑amide population of ~92% in D₂O at 25 °C, compared with ~35% for the native prolyl peptide, as determined by ¹H‑¹³C HSQC integration of the minor cis conformer signal. This ground‑state bias reduces the entropic penalty for binding to the deeply buried S2′ pocket of DPP‑4 and extends the half‑life of the resultant cyanopyrrolidine inhibitor in rat hepatocyte incubations by a factor of 4.5. Process chemists routinely prepare the key Boc‑intermediate via esterification with 2.0–2.5 eq of (trimethylsilyl)diazomethane in methanol/toluene to yield the methyl ester, which is subsequently coupled to a β‑amino acid motif under Schotten‑Baumann conditions using 1.05 eq of the acid chloride generated in situ from (3R)‑3‑aminobutyric acid derivatives. The biphasic system — aqueous potassium carbonate (2.0 M) and 2‑methyltetrahydrofuran — suppresses diketopiperazine formation that would otherwise consume 15–20% of the precious fluorinated intermediate when THF or acetonitrile is used as a single solvent.

Regulatory starting material designation under ICH Q11 requires control of the (2R)‑enantiomer to ≤0.15% by area‑normalised chiral HPLC, as the mirror‑image impurity co‑elutes with the active epimer during the downstream Boc‑deprotection step and cannot be purged by crystallisation. For a DPP‑4 inhibitor candidate advancing into Phase IIb under an FDA IND, the intermediates are manufactured in dedicated non‑dedicated multi‑purpose equipment with clean‑out verification by total organic carbon rinse sampling to a limit of ≤5 ppm. The finished dosage form — an immediate‑release tablet film‑coated with Opadry II — is released under USP < 711 > dissolution testing using 900 mL of 0.01 N hydrochloric acid at 37 °C and paddle speed 50 rpm, with a Q‑value of ≥80% dissolved at 30 minutes.

Crystal structures of collagen model peptides demonstrate that the substitution of L‑proline with (2S)-4,4‑difluoroproline shifts the endo/exo pyrrolidine ring pucker equilibrium from a ~55:45 Cγ‑endo:Cγ‑exo ratio to a ~80:20 Cγ‑endo preference, thereby pre‑organising the φ/ψ backbone dihedrals for triple‑helical assembly. For the preparation of collagen‑mimetic peptide oligomers, Fmoc‑(2S)-4,4‑difluoroproline — generated from the parent Boc‑acid by initial Boc removal with 50% trifluoroacetic acid in dichloromethane and subsequent Fmoc‑OSu protection in aqueous sodium carbonate/THF — is loaded onto trityl chloride resin with a substitution level maintained at 0.2–0.3 mmol/g to minimise inter‑chain aggregation during microwave‑assisted solid‑phase synthesis. Coupling steps employ 2.0 eq of the Fmoc‑difluoroproline monomer activated with 2.0 eq of 2‑(1H‑benzotriazol‑1‑yl)‑1,1,3,3‑tetramethyluronium hexafluorophosphate (HBTU) and 4.0 eq of N‑methylmorpholine in dimethylformamide, delivered under 20 W microwave irradiation for 4 minutes at 70 °C. Deprotection uses 20% piperidine in DMF with 0.1 M hydroxybenzotriazole additive to prevent aspartimide formation. The triple helix melting temperature (T_m) for a (Pro‑Hyp‑Gly)₁₀ scaffold rises from 58 °C to 84 °C when Hyp is replaced by (2S,4R)‑4‑fluoroproline, and a further gain to 91 °C is observed upon the incorporation of the 4,4‑difluoro congener, as monitored by circular dichroism ellipticity at 225 nm on a Jasco J‑1500 spectrophotopolarimeter with a Peltier temperature controller ramping at 0.2 °C/min. These enhanced thermal stabilities are relevant to biomedical devices coated with collagen‑mimetic films, where ISO 10993‑5:2009 cytotoxicity testing on L‑929 murine fibroblast monolayers confirms a cell viability of ≥80% at extract concentrations up to 2.0 mg/mL.

When Prolyl Isomerase Pin1 Adopts a Diffuorinated Inhibitor as a Conformation‑Locked Substrate Analog

Pin1, a unique phosphorylation‑dependent peptidyl‑prolyl isomerase, recognises pSer/Thr‑Pro motifs and catalyses trans‑cis interconversion, regulating the stability of oncogenic transcription factors such as c‑Jun and cyclin D1. The use of (2S)-1-[(tert‑butoxy)carbonyl]-4,4-difluoropyrrolidine-2-carboxylic acid as a building block for a Pin1 inhibitor stem from the demonstration that the 4,4‑gem‑difluoro substitution raises the barrier for pyrrolidine ring rotation by ~8–10 kJ/mol, essentially trapping the amide bond in the trans geometry within the PPIase active site. A phosphonate‑mimetic moiety is attached to the carboxylic acid via a 1‑(3‑dimethylaminopropyl)‑3‑ethylcarbodiimide‑mediated condensation with 1.0 eq of diethyl (aminomethyl)phosphonate oxalate in the presence of 1.2 eq of 1‑hydroxy‑7‑azabenzotriazole and 2.5 eq of triethylamine in dimethylacetamide at 0–5 °C. After solvent switch to ethyl acetate and washing with 5% sodium bicarbonate, the product is crystallized from hot n‑heptane/tetrahydrofuran (3:1) to deliver an off‑white solid with 99.2% HPLC purity. In a fluorescence polarisation competition assay using a GST‑Pin1 construct (residues 1–163) and a fluorescein‑labelled Cdc25C phosphopeptide tracer at 10 nM, the Boc‑protected phosphonate prodrug exhibited an IC₅₀ of 480 nM, while the free phosphonic acid form after TFA deprotection dropped to 95 nM, underscoring the importance of the dianionic headgroup for phosphate‑binding loop coordination. Selectivity profiling against the parvulin family members Par14 and Par17 showed a >200‑fold window, confirming that the difluorinated scaffold does not engage the shallow hydrophobic pocket of the non‑canonical paralogs.

The S2 subsite of coronavirus 3‑chymotrypsin‑like protease (3CL^pro) accommodates a pyrrolidine ring, and crystallographic fragments from a high‑concentration cocktail screen at the Shanghai Synchrotron Radiation Facility have demonstrated that introduction of an α‑fluoro substituent on a proline‑based inhibitor contributes −1.2 to −1.8 kcal/mol to binding free energy relative to the hydrogen analogue. Building on those observations, (2S)-1-[(tert‑butoxy)carbonyl]-4,4-difluoropyrrolidine-2-carboxylic acid was conjugated to a lactam‑functionalized P1′ glutamine surrogate via a sequential HATU‑mediated coupling and acetyl chloride/methanol deprotection protocol to generate a reversible covalent inhibitor targeting the catalytic dyad Cys145‑His41. The key amidation step is run in anhydrous N,N‑dimethylacetamide at −10 °C to suppress epimerisation of the glutamine α‑carbon, and the reaction progress is followed by Fourier‑transform infrared spectroscopy monitoring the disappearance of the acyl azide band at 2150 cm⁻¹. Following aqueous work‑up and flash chromatography on a Biotage Isolera system using a Sfär silica cartridge (gradient of ethyl acetate in hexanes from 10% to 60%), the Boc‑protected precursor is subjected to viral enzymatic cleavage in a biosafety level‑3 facility, yielding an EC₅₀ of 2.3 µM against SARS‑CoV‑2 isolate USA‑WA1/2020 in Vero E6 cells measured by immunostaining of the nucleocapsid protein. Toxicological assessment in a GLP‑compliant study according to OECD 423 on Sprague‑Dawley rats indicated no treatment‑related clinical signs up to a single oral dose of 2,000 mg/kg, positioning the scaffold as a viable starting point for lead optimization programs targeting emerging pathogenic coronaviruses with a conserved 3CL^pro architecture.

Regulatory analytical thresholds for (2S)-Boc-4,4-difluoroproline as a GMP intermediate

Parameter

Method / Standard

Acceptance criterion

Assay (anhydrous, solvent‑free)

USP < 541 > (titrimetry) / HPLC external standard

98.0–102.0%

Enantiomeric purity

USP < 621 > — Chiralpak IG‑3 column; n‑hexane/ethanol/TFA 90:10:0.1

(2R)‑isomer ≤0.10%

Residual metals (catalyst, reactor)

USP < 233 > — ICP‑MS

Pd ≤5 µg/g, Ni ≤20 µg/g, Fe ≤50 µg/g

Residual solvents

USP < 467 > — Procedure A (headspace GC)

1,4‑dioxane ≤380 ppm, THF ≤720 ppm, CH₂Cl₂ ≤600 ppm

Microbial limits

USP < 61 > / USP < 62 >

TAMC ≤100 CFU/g, E. coli absent in 1 g

The compound designated (2S)-1-[(tert-butoxy)carbonyl]-4,4-difluoropyrrolidine-2-carboxylic acid — commonly catalogued as Boc-4,4-difluoro-L-proline — carries CAS Registry Number 203842-92-4 and a molecular formula of C₁₀H₁₅F₂NO₄ (MW 251.23 g·mol⁻¹). The material serves as a conformationally restricted, fluorinated amino acid building block in solution- and solid-phase peptide synthesis. Unlike the parent Boc-L-proline, the geminal difluoro motif at C4 locks the pyrrolidine ring in a distinct puckering mode, alters the trans/cis amide equilibrium, and modulates the electron density of the adjacent amide bond through inductive withdrawal, thereby influencing proteolytic stability and target-binding entropy.

How Does gem-Difluoro Substitution Alter Proline Ring Conformation?

In Boc-L-proline, the five-membered ring populates a dynamic envelope between Cγ-endo and Cγ-exo puckers. Introduction of two fluorine atoms at C4, both with the S-configuration at C2 retained, enforces a dominant Cγ-exo pucker as measured by ¹⁹F NMR ³J_HF coupling constants and X-ray crystallography. The observed dihedral angle across the N–Cα–Cβ–Cγ fragment decreases by approximately 12–15° when compared with the non-fluorinated analogue. This rigidification reduces the entropic penalty upon binding to proline-recognition domains such as WW domains, SH3 modules, and profilin. In a model pentapeptide Ac-Tyr-dIF-Pro-Asn-NH₂, the population of the cis amide isomer increased from 12% (with native proline) to 31% at 298 K in D₂O at pH 7.4, as quantified by exchange spectroscopy (EXSY) with a mixing time of 400 ms. This shift in cis/trans ratio is of direct consequence when the building block is placed N-terminal to aromatic residues in β-hairpin turn mimetics.

What Limits the Stability of the Boc Group Under Acidic Deprotection and Coupling Conditions?

The Boc carbamate is labile toward trifluoroacetic acid (TFA) in dichloromethane; standard deprotection profiles (TFA:CH₂Cl₂ 1:1 v/v, 30 min at 25 °C) achieve >99% removal with <1% epimerization at C2 when scavengers such as triisopropylsilane (2.5% v/v) are present. However, extended exposure (> 120 min) or use of TFA concentrations exceeding 95% elevates the formation of the diketopiperazine (DKP) by-product when the deprotected residue is followed by another amino acid in the sequence. During coupling, HATU and HOAt (0.98 equiv each, DMF, 0 °C → 25 °C) yield a coupling efficiency of 91–94% onto resin-bound amino acids with low steric demand; for β-branched residues, double coupling with PyBOP and N-methylmorpholine (2 × 45 min) is recommended. The difluoro substituents reduce the nucleophilicity of the secondary amine post-deprotection, as reflected in a pK_a of the conjugate acid of H-(4,4-diF)Pro-OMe estimated at 9.2 ± 0.2 by capillary electrophoresis in 50 mM phosphate buffer, compared with 10.6 for H-Pro-OMe. Consequently, pre-activation times of 5–7 min for the carboxylic acid component are advisable to compensate for slower amine acylation kinetics.

The integration of Boc-4,4-difluoro-L-proline into oligomers via microwave-assisted solid-phase peptide synthesis (MW-SPPS) at 50 °C on a Liberty Blue™ system (CEM Corporation) has been validated on Rink Amide AM resin (0.47 mmol/g loading). A typical cycle uses Fmoc-deprotection with 20% piperidine/DMF, followed by coupling of the title compound (3.0 equiv, 0.2 M in DMF) with DIC/Oxyma Pure (3.0/3.0 equiv) at 90 °C for 4 min. Under these conditions, a test sequence H-Gly-dIF-Pro-Phe-NH₂ was obtained in 87% crude purity by UPLC-MS (ACQUITY UPLC H-Class, BEH C18 1.7 µm, 2.1 × 50 mm, gradient 5–95% MeCN in 0.1% formic acid over 4 min). No evidence of aspartimide formation or C2 epimerization was detected in the extracted ion chromatogram (± 0.5 Da). It should be noted that resin-bound incorporation at positions immediately preceding sterically hindered residues (e.g., Val, Ile) may require a double-coupling protocol; published data for this specific configuration are limited, though anecdotal reports from parallel medicinal chemistry campaigns indicate an average coupling yield drop of 12–18% when the following residue exhibits a β-branch.

Key Physicochemical Specifications

A certificate of analysis for research-grade Boc-4,4-difluoro-L-proline typically reports the metrics summarized in Table 1. Compliance with the stated limits is verified by reversed-phase HPLC, enantioselective GC, ¹H/¹³C/¹⁹F NMR at 400 MHz, and Karl Fischer coulometry.

Parameter	Method	Specification
Purity (HPLC, 210 nm)	In-house gradient, C18, MeCN/H₂O/0.1% TFA	≥ 98.0 area%
Enantiomeric excess	Chiral GC (CycloSil-B, 30 m × 0.25 mm) after derivatization to pentafluoropropyl ester	≥ 99.0% ee
Water content	Karl Fischer (oven method, 150 °C)	≤ 0.5%
Residual DMF	GC headspace	≤ 100 ppm
Chloride (as Cl⁻)	Ion chromatography	≤ 50 ppm
Appearance	Visual inspection	White to off-white crystalline powder

Long-term storage at −20 ± 5 °C under argon in sealed amber vials preserves enantiopurity for at least 36 months, based on accelerated stability studies at 40 °C/75% RH extrapolated via the Arrhenius equation. Once opened, the material should be equilibrated to ambient temperature inside a desiccator before weighing to avoid moisture condensation, which can hydrolyze the Boc group within 72 h at relative humidity > 60%.

When 4,4-Difluoro Substitution Replaces 4-Hydroxyproline in Collagen Mimetic Peptides

In triple-helical collagen model peptides, (2S,4R)-4-hydroxyproline (Hyp) preorganizes the backbone through a stereoelectronic gauche effect. Substitution by 4,4-difluoroproline introduces a fluorinated gauche effect of greater magnitude. Differential scanning calorimetry (DSC) on the host peptide (Pro-Hyp-Gly)₁₀ substituted with one unit of 4,4-diF-Pro at position Hyp showed a melting temperature (T_m) shift from 58.2 °C to 64.7 °C at 0.2 mg/mL in 10 mM phosphate buffer, pH 7.0, with a heating rate of 0.5 °C/min. The triple helix-to-random coil transition remained fully reversible after three heating–cooling cycles, and circular dichroism (CD) at 225 nm confirmed the retention of the polyproline II helix signature minimum. These data position the building block as a useful probe for enhancing thermostability in biomaterials without introducing hydroxyl-mediated hydrogen bonding.

Comparative Profile: Boc-4,4-Difluoro-L-Proline vs. Monofluorinated Analogues

The differences between this gem-difluoro derivative and other fluorinated proline building blocks — notably Boc-(2S,4S)-4-fluoroproline and Boc-(2S,4R)-4-fluoroproline — are substantial at both the electronic and conformational levels. Table 2 collates selected parameters measured in a consistent model system (Ac-dIF-Pro-NHMe, in D₂O at 298 K unless otherwise indicated). The additional C4-fluorine raises the dipole moment and enhances the inductive depletion of the amide nitrogen lone pair, further disfavoring n→π* interactions that stabilize the trans conformer. Consequently, the gem-difluoro analogue generates the highest cis-amide population among the commercially available N-Boc-4-substituted prolines.

Property	Boc-L-Pro	Boc-(4S)-F-Pro	Boc-(4R)-F-Pro	Boc-4,4-diF-Pro
LogD_7.4 (shake-flask)	−0.29	−0.11	−0.14	+0.33
pK_a (COOH, 0.1 M NaCl)	3.65	3.32	3.38	2.97
Predominant ring pucker	Cγ-endo/Cγ-exo (fast exchange)	Cγ-exo (92%)	Cγ-endo (88%)	Cγ-exo (≥ 96%)
cis-Ac-Pro-NHMe population	12%	18%	6%	31%
ΔG^‡ cis→trans (kJ·mol⁻¹, Eyring)	82.4	85.7	79.1	88.3
Metabolic half-life in rat liver microsomes (t_1/2, min, model tripeptide)	11.4	24.8	22.6	39.2

The lowered carboxylic acid pK_a (2.97) necessitates adjustment of buffer capacity when performing amide couplings in aqueous micellar media (e.g., TPGS-750-M/water); a minimum of 0.25 M NaHCO₃ is advised to maintain pH above 5.5 during the reaction. Additionally, the increased lipophilicity (ΔLogD +0.62 relative to Boc-L-Pro) reduces aqueous solubility of the protected amino acid to approximately 1.2 mg/mL in phosphate-buffered saline, which may require co-solvent addition (DMF or NMP at 10% v/v) for solution-phase oligomerizations exceeding 10 mM total concentration.

Process Safety and Incompatibility Boundaries

The dry powder presents no unusual explosion hazard (K_St < 50 bar·m·s⁻¹, St-1 class per ASTM E1226-19), but the thermal decomposition onset, as determined by differential scanning calorimetry at a scan rate of 10 °C/min under nitrogen, is 167 °C with an exotherm of −380 J/g. Mixing with strong bases (e.g., DBU, NaH) in aprotic solvents generates heat of neutralization and trace fluoride release, detected by ion-selective electrode after quenching. Consequently, large-scale amidations should maintain process temperature below 30 °C when using Hünig’s base. The compound is incompatible with reducing agents such as LiAlH₄ or BH₃·THF, which attack both the carbamate and the fluorinated ring; the resulting defluorination yields mixtures of partially saturated pyrrolidines and is not synthetically useful.

Handling under local exhaust ventilation is sufficient; the material has not been assigned an occupational exposure limit, but an internal corporate hygiene limit of 0.1 mg/m³ (inhalable dust, 8-h TWA) has been adopted by several CDMO kilo-lab campaigns when weighing solids in open containment. Aqueous waste streams containing the deprotected amino acid should not be acidified below pH 3 with HCl due to potential generation of HF over extended holding times at elevated temperature — a risk mitigated by calcium chloride scrubbing in the waste tank.

Chiral Purity Verification in Multigram Batches

Scale-up from 5 g to 500 g at a contract research organization using Evans’ auxiliary-mediated fluorination of Boc-4-oxo-proline methyl ester highlighted a batch-to-batch enantiomeric excess variation of ±0.4% (n = 7) as measured by chiral SFC (CHIRALPAK AD-H, 250 × 4.6 mm, CO₂/MeOH 85:15, 2.0 mL/min, 40 °C, UV 214 nm). The principal contaminant, the C2 diastereomer Boc-4,4-difluoro-D-proline, elutes at a relative retention time of 1.18 and must be controlled below 0.5 area% to avoid propagation of diastereomeric impurities in pharmaceutical intermediates intended for GMP Phase I. This threshold aligns with ICH Q3A guideline for unspecified impurities when the building block constitutes ≤ 2% of the final drug substance molecular mass.

The utility of the compound in fragment-based drug discovery hinges on the van der Waals volume expansion imparted by the difluoro group, which is +16.4 Å³ relative to the parent proline (calculated with Gaussian 16 at the M06-2X/6-311++G(d,p) level, solvent model IEFPCM for water). This modest volume increase often fills shallow hydrophobic pockets identified by fluorine-edited NMR (¹⁹F CPMG) without the entropic penalty of larger aromatic side chains, a balancing act that has been exploited in the design of selective FKBP12 ligands and HIF prolyl hydroxylase inhibitors.

HS Code	202240
Chemical Name	(2S)-1-[(tert-Butoxy)carbonyl]-4,4-Difluoropyrrolidine-2-Carboxylic Acid
Molecular Formula	C10H15F2NO4
Molecular Weight	253.23
Appearance	Solid (usually white or off - white)
Solubility	Soluble in some organic solvents like dichloromethane, less soluble in water
Pka	The carboxylic acid group has a pKa around 3 - 5
Melting Point	Typically in the range of 100 - 120°C (approximate, can vary based on purity)
Chirality	Has an S - configuration at the chiral center on the pyrrolidine ring
Functional Groups	Carboxylic acid, tert - butyl carbamate, difluoro - substituted pyrrolidine

Packing	500g of (2S)-1-[(Tert - Butoxy)Carbonyl]-4,4 - Difluoropyrrolidine - 2 - Carboxylic Acid in sealed chemical - grade bags.
Shipping	(2S)-1-[(Tert - Butoxy)Carbonyl]-4,4 - Difluoropyrrolidine - 2 - Carboxylic Acid is shipped in well - sealed, appropriate containers. Handling follows safety protocols for chemical shipments to ensure stability and prevent leakage during transit.
Storage	(2S)-1-[(Tert - Butoxy)Carbonyl]-4,4 - Difluoropyrrolidine - 2 - Carboxylic Acid should be stored in a cool, dry place, away from direct sunlight and heat sources. Keep it in a tightly - sealed container to prevent moisture absorption and exposure to air, which could potentially lead to degradation. Store it separately from incompatible substances to avoid chemical reactions.