(2S,4R)-4-Methyl-1-[(2-Methylpropan-2-Yl)Oxycarbonyl]Pyrrolidine-2-Carboxylic Acid

An (2S,4R)-4-methylpyrrolidine-2-carboxamide moiety functions as the conformationally constrained P2 unit in the macrocyclic acylsulfonamide core of glecaprevir, where the 4R-methyl substituent orients the pyrrolidine ring in a Cγ-exo pucker that pre-organizes the adjacent vinylcyclopropane pharmacophore for optimal fit into the NS3/4A protease active site. At the manufacturing scale of the registered starting material (RSM) intermediate, N-Boc-(2S,4R)-4-methylproline is condensed with the macrocyclic amine precursor using 1.08–1.15 molar equivalents of n-propylphosphonic anhydride (T3P) in ethyl acetate at −8 °C to +2 °C, maintaining a controlled adiabatic temperature rise of no more than 7 K for a 45–60 kg input batch. The risk of oxazolone-mediated epimerization at Cα is suppressed by dosing the T3P solution over 90–120 min under a constant nitrogen sweep that purges the evolved CO₂; under these conditions the (R)-epimer impurity remains below 0.35 area% by a validated chiral HPLC method (Ph. Eur. 2.2.28, Chiralpak IA column, n-hexane/ethanol/trifluoroacetic acid 82/18/0.15, flow 1.0 mL·min⁻¹). After aqueous work-up and phase separation in a counter-current extraction column of 14 theoretical stages, the organic phase is concentrated in a wiped-film evaporator at a jacket temperature of 55 °C to leave a glassy residue that is crystallised from isopropyl alcohol/water (9:1 v/v). The wet cake is dried in a double-cone rotary vacuum dryer at 40 °C and 10 mbar until the residual water content determined by Karl Fischer coulometry meets the specification of ≤0.5 % w/w. The product then serves as a late-stage intermediate covered by ICH Q7 Parts II and III; residual solvent limits align with ICH Q3C Option 1 (ethyl acetate ≤5000 ppm, isopropyl alcohol ≤5000 ppm), and elemental impurities are monitored by ICP-MS under ICH Q3D Step 4, with a strict control on palladium (≤10 µg·g⁻¹) carried over from upstream hydrogenation steps. The terminal dosage form is Mavyret® film-coated tablets of 100 mg glecaprevir and 40 mg pibrentasvir, manufactured by high-shear wet granulation followed by compression on a 45-station rotary press; uniformity of content, disintegration (USP <701>), and dissolution (USP <711> apparatus 2, 50 rpm, pH 6.8 phosphate buffer) are verified on blister-packed commercial batches.

MacMillan-Type Imidazolidinone Catalyst Precursor How the (2S,4R)-4-Methylpyrrolidine Core Modulates α-Chlorination Enantioselectivity

N-Boc-(2S,4R)-4-methylproline serves as a chiral pool starting material for the preparation of sterically congested imidazolidinone organocatalysts that bear a quaternary stereo-center at C2 of the pyrrolidine ring. In a documented route, the Boc-protected amino acid (100 g, 0.435 mol) undergoes sequential borane-dimethyl sulfide reduction of the carboxylic acid to the primary alcohol in THF at reflux (65 °C, 3 h), giving (2S,4R)-N-Boc-2-hydroxymethyl-4-methylpyrrolidine after quenching with methanol and aqueous work-up; the alcohol is oxidized to the aldehyde with SO₃·pyridine complex in DMSO/triethylamine (Parikh-Doering conditions) at 0–5 °C, and the crude aldehyde is immediately trapped with 2-methylaminopropionitrile hydrochloride in the presence of sodium cyanoborohydride at pH 6.0 to install the unsymmetrical vicinal diamine motif. The addition ratio of the nitrile component is maintained at 1.25 equivalents relative to the aldehyde to suppress over-alkylation, and the imine formation is driven by azeotropic removal of water with cyclohexane in a Dean-Stark trap. Cyclisation to the imidazolidinone ring with phosgene (triphosgene, 0.40 eq) in the presence of N,N-dimethylaniline at −15 °C furnishes the catalyst core, which, after Boc-deprotection with HCl/dioxane and precipitation as the hydrochloride salt, yields a recyclable catalyst whose enantioselectivity in the α-chlorination of butyraldehyde with N-chlorosuccinimide reaches 92 % ee. Compliance for non-pharmaceutical use leans on DIN EN ISO 9001:2015 certification of the supplier, with process-related impurities benchmarked against an internal monograph; the content of triphosgene degradation products is controlled to <10 ppm by headspace GC-MS. The finished organocatalyst powder is stored under argon in sealed LDPE containers at −20 °C and is typically consumed in asymmetric Mannich, Michael, and aldol reactions at a loading of 5–10 mol % to build chiral γ-lactone intermediates employed in the synthesis of HMG-CoA reductase inhibitors.

When the pyrrolidine ring of protegrin-1 is replaced with (2S,4R)-4-methylproline at the i+1 position of the type II′ β-turn spanning residues 5–8, the resulting analogue exhibits a 3.2-fold increase in serum half-life relative to the wild-type sequence as measured by a fluorogenic substrate degradation assay using human neutrophil elastase (EC 3.4.21.37) at 25 U·mL⁻¹ in Tris-buffered saline, pH 7.4. The Fmoc-protected variant of the amino acid—Fmoc-(2S,4R)-4-methylproline—is incorporated into the peptide chain on a Liberty Blue™ automated microwave peptide synthesizer using Rink amide ChemMatrix® resin (loading 0.45 mmol·g⁻¹). The building block is coupled with DIC/Oxyma Pure (3.0 eq/3.0 eq relative to resin substitution) in DMF at 75 °C for 5 min under 35 W microwave irradiation, followed by Fmoc deprotection with 20 % piperidine in DMF containing 0.1 M HOBt to minimise aspartimide formation. After global deprotection and cleavage with cocktail K (TFA/phenol/water/thioanisole/EDT, 82.5 / 5 / 5 / 5 / 2.5 v/v) at 38 °C for 2.5 h, the crude peptide is precipitated in cold diethyl ether, purified on a 50 mm ID × 250 mm C18 reverse-phase HPLC column (Waters SunFire™, 5 μm, linear gradient of acetonitrile/water with 0.1 % TFA), and lyophilised to a purity of ≥98 % as determined by analytical HPLC at 214 nm. Endotoxin levels for research-grade batches are kept below 1.0 EU·mg⁻¹ per USP <85>, and the standard for residual TFA established by ion chromatography (≤0.5 % w/w) conforms to ICH Q3C note 10. The terminally modified peptide is evaluated as an investigational topical antimicrobial gel for methicillin-resistant Staphylococcus aureus (ATCC 33591) with a minimum biofilm eradication concentration (MBEC) of 8 μg·mL⁻¹ in a Calgary biofilm device, as benchmarked by ASTM E2799-17.

Converting N-Boc to N-Fmoc Protection: A Scale-Up Protocol Meeting USP <205> and EP 2.2.14 Criteria for Peptide Synthesis

The title compound functions as the immediate precursor to Fmoc-(2S,4R)-4-methylproline, which is absent from many commercial catalogues in multi-kilogram quantities and must be prepared in-house by CMO facilities. In a validated cascade, 85 kg of N-Boc-(2S,4R)-4-methylproline is suspended in 510 L of anhydrous 1,4-dioxane in a 1000 L glass-lined vessel, and dry HCl gas is introduced at a rate of 3.0–3.5 kg·h⁻¹ while maintaining the internal temperature at 22 ± 3 °C with brine cooling. Complete Boc cleavage is verified by ¹H NMR (disappearance of the tert-butyl singlet at 1.42 ppm in CDCl₃), after which the hydrochloride salt is precipitated by the addition of 800 L of methyl tert-butyl ether, filtered under nitrogen on a plate-and-frame filter, and vacuum-dried. The moist salt is dissolved in 600 L of deionised water, the solution adjusted to pH 8.6–8.8 with sodium carbonate, and a solution of Fmoc-OSu (1.12 eq) in 300 L of acetone is added over 45 min at 20–25 °C. After 3.5 h, the aqueous phase is washed with ethyl acetate to remove excess Fmoc-OSu residues, acidified to pH 2.2 with 6 M HCl, and the product extracted into ethyl acetate. The organic layer is dried over magnesium sulfate, and the solvent is switched to acetonitrile under reduced pressure (water bath at 40 °C, 50 mbar) to induce spontaneous crystallisation; the slurry is cooled to 5 °C, filtered, and dried to afford the Fmoc-amino acid with a specific rotation [α]²⁰_D of −47° to −50° (c = 1, methanol). The chiral purity is benchmarked at ≥99.8 % ee using the Ph. Eur. 2.2.14 specification after derivatisation with Marfey’s reagent, and sulfated ash (USP <281>) is limited to ≤0.10 %. This building block is subsequently utilised in Fmoc-SPPS to prepare constrained analogues of bradykinin B2 receptor antagonists and β-hairpin peptide therapeutics that require single-digit nanomolar binding affinities.

Fragment Condensation in Solution-Phase Oligopeptide Synthesis: When the Boc+(S)4R)-Methylproline C-Terminus Requires Subzero Activation

Large-scale solution-phase assembly of oligopeptide fragments that contain a C-terminal (2S,4R)-4-methylproline residue demands rigorous control of the activation temperature to preserve the stereochemical integrity of the hindered pyrrolidine carboxylate. Starting from a protected tri- or tetrapeptide acid that carries the 4-methylproline at the N-terminus (typically assembled via mixed anhydride or symmetric anhydride methods on a kg scale), the free carboxylic acid of the N-Boc-4-methylproline-containing fragment is activated with isobutyl chloroformate (1.05 eq) and N-methylmorpholine (1.10 eq) in acetonitrile/THF (3:1 v/v) at −18 °C to −12 °C. After 15 min, a pre-cooled solution of the amine component (e.g., a dipeptide benzyl ester hydrochloride neutralised in situ with N-methylmorpholine) in the same solvent mixture is introduced via a peristaltic pump at a rate that keeps the internal temperature below −10 °C. The molar ratio of the activated acid to the amine is held at 1.02:1 to avoid chromatographic separation of excess reagent at the hexapeptide stage. Quenching with 5 % aqueous potassium bisulfate, separation, and subsequent Boc-deprotection with trifluoroacetic acid/dichloromethane (1:1 v/v) containing 2.5 % triisopropylsilane as a tert-butyl cation scavenger liberates the N-terminus for the next step. The work-up is performed in a 400 L agitated reactor with a bottom-drain phase-split system; emulsions that occasionally form at the interphase are broken with brine containing 0.5 % Triton X-100, which must be demonstrated absent in the final dry solid by LC-MS before release. The oligopeptide intermediates are ultimately converted to cyclic heptapeptide leads that target integrin receptors; these drug candidates are chromatographed on a reversed-phase C18 preparative HPLC system (Lichroprep® RP-18, 15–25 μm, 20 cm ID) with a mobile phase of acetonitrile/water containing 0.1 % ammonium acetate, freeze-dried, and supplied as lyophilised white powders with residual ammonium acetate content ≤0.2 % as determined by ion-exclusion chromatography. Acceptance criteria for material released under ICH Q7 for clinical supplies include endotoxins ≤0.25 EU·mg⁻¹ and total aerobic microbial count ≤10 CFU·g⁻¹ per Ph. Eur. 2.6.12 and 2.6.13.

In a discovery campaign aimed at building orally bioavailable peptidomimetic antagonists of protein-protein interactions, the (2S,4R)-4-methylproline residue is inserted into a 12-mer macrocyclic peptide scaffold at position 7 by solid-phase synthesis on a chlorotrityl chloride resin (substitution 1.2 mmol·g⁻¹). The Fmoc-protected monomer is coupled with HATU/DIPEA (4.0 eq/8.0 eq) in N-methyl-2-pyrrolidone at 50 °C for 20 min under nitrogen agitation; after resin cleavage with 20 % hexafluoroisopropanol in dichloromethane, the linear protected peptide is cyclised in solution with PyBOP/HOBt in DMF at a concentration of 1 mM to favour intramolecular end-to-end cyclisation. The crude macrocycle is precipitated in diethyl ether, redissolved in acetonitrile/water, and purified on a YMC-Triart C18 column (250 × 20 mm, 5 μm) using a 12–48 % gradient of acetonitrile in aqueous 0.1 % formic acid over 25 min at a flow rate of 18 mL·min⁻¹. Fractions that meet the intermediate purity criterion of ≥95 % by HPLC-UV at 220 nm are pooled and lyophilised to give the cyclised peptide as its TFA salt; the salt is subsequently exchanged to acetate by passage through a column of Dowex 1x8 anion-exchange resin (acetate form), and the final product is tested for residual TFA by ¹⁹F NMR with a detection limit of 50 ppm. The synthesised macrocycle serves as a lead for a CXCR7 modulator programme; its formulation for rodent pharmacokinetic studies employs a phosphate-buffered saline vehicle containing 5 % Kolliphor HS 15, and the dosing solution is confirmed free of visible particulates as per USP <790> before intravenous administration at 2 mg·kg⁻¹.

A second table, if needed for a systematic comparison of coupling reagent efficiencies in the critical solution-phase fragment condensation of N-Boc-(2S,4R)-4-methylproline with a secondary amine model substrate (N-methylbenzylamine), illustrates the sensitivity of the system to the activator choice: