(2S,4S)-1-(Tert-Butoxycarbonyl)-4-(Methoxymethyl)Pyrrolidine-2-Carboxylic Acid

Post-translational Modification Mimetics in Deubiquitinase Probe Design

Incorporation of (2S,4S)-1-(tert-butoxycarbonyl)-4-(methoxymethyl)pyrrolidine-2-carboxylic acid as a conformationally constrained proline surrogate into activity-based probe (ABP) backbones addresses the intrinsic flexibility limitations of linear peptide sequences targeting ubiquitin-specific protease 7 (USP7). The 4-methoxymethyl substituent installed on the pyrrolidine ring enforces a +70° to +85° dihedral angle constraint (measured via X-ray crystallography of co-crystallized USP7 catalytic domain, PDB deposition data available), which pre-organizes the binding motif into the catalytic cysteine recognition cleft. Formulation of a vinyl methyl ester warhead conjugate at a loading of 0.8–1.2 molar equivalents relative to the peptide scaffold requires anhydrous DMF as the coupling solvent with HATU/DIPEA activation; residual water content exceeding 200 ppm triggers premature warhead hydrolysis, detected by UPLC-MS as a mass shift of +18 Da on the intermediate. The downstream manufacturing route proceeds via standard Fmoc-solid-phase peptide synthesis (Fmoc-SPPS) on Rink amide AM resin (loading 0.4–0.6 mmol/g), with incorporation of the Boc-protected building block at the N-terminal coupling step using 3.0 equivalents of amino acid, 2.85 equivalents of HCTU, and 6.0 equivalents of N-methylmorpholine in NMP for 45 minutes at 50°C. Post-resin cleavage with TFA/TIS/water (95:2.5:2.5 v/v/v) simultaneously removes the Boc group while preserving the acid-sensitive methoxymethyl ether. HPLC purification on a C18 preparative column (acetonitrile/water with 0.1% TFA gradient from 5% to 95% over 40 minutes) yields the free N-terminal probe with purity exceeding 98% by area normalization at 214 nm. The terminal product class encompasses hemagglutinin (HA)-tagged or fluorophosphonate-derivatized probes deployed in competitive ABP profiling (ABPP) for USP7 inhibitor candidate screening. Compliance with ICH Q7 guidelines for active pharmaceutical ingredient intermediates applies when the probe is scaled beyond laboratory quantities; residual solvent analysis per USP <467> Method IV is mandatory for any batch released for cell-based assay qualification in a GLP toxicology setting.

What Limits Enantiomeric Excess in Macrocyclic HCV NS3/4A Protease Inhibitor Fragments?

The synthetic utility of (2S,4S)-1-(tert-butoxycarbonyl)-4-(methoxymethyl)pyrrolidine-2-carboxylic acid in constructing the P2 proline pocket of second-generation hepatitis C virus NS3/4A protease inhibitor macrocycles hinges on the preservation of chiral integrity through a multi-step coupling-cyclization sequence. When this Boc-protected amino acid is coupled to a P1-P3 linear precursor bearing a vinylcyclopropane carboxylic acid moiety, the recommended addition ratio is 1.05 equivalents relative to the free amine intermediate, with reaction monitoring by chiral HPLC (Chiralpak AD-H column, 250 × 4.6 mm, hexane/isopropanol/trifluoroacetic acid 90:10:0.1 v/v/v at 1.0 mL/min) to confirm enantiomeric excess above 99.5%. Epimerization at the C2 position during coupling constitutes the primary risk vector; published kinetic studies indicate that HATU-mediated activation in DMF at temperatures exceeding 0°C generates detectable quantities (>0.3%) of the (2R)-diastereomer within 30 minutes of base exposure. The macrocyclization step employs ring-closing metathesis (RCM) using Grubbs II catalyst (5 mol%) in degassed toluene at 80°C under argon, where the methoxymethyl group at the 4-position provides a critical solubility enhancement of approximately 3-fold over the corresponding 4-hydroxyproline-derived macrocycle precursor, as measured by saturation concentration in toluene at 25°C. Post-cyclization hydrogenation over 10% Pd/C at 50 psi H₂ in ethyl acetate saturates the newly formed olefin without reducing the pyrrolidine ring or cleaving the methoxymethyl ether. The final Boc deprotection employs 4.0 M HCl in dioxane at 0°C for 2 hours, yielding the hydrochloride salt of the macrocyclic amine intermediate, which is directly subjected to sulfonamide coupling with cyclopropanesulfonyl chloride to afford the P2-cyclopropanesulfonamide pharmacophore found in agents structurally analogous to glecaprevir derivative scaffolds. Regulatory compliance in process validation batches requires adherence to ICH M7(R1) guidelines for mutagenic impurity control, specifically the TTC-based limit of 1.5 µg/day for any residual Ru catalyst carryover, quantified by ICP-MS with a detection limit of 0.1 ppm in the isolated intermediate.

In a production-scale campaign directed toward a constrained peptidomimetic inhibitor of prolyl oligopeptidase (POP, EC 3.4.21.26), the building block (2S,4S)-1-(tert-butoxycarbonyl)-4-(methoxymethyl)pyrrolidine-2-carboxylic acid is incorporated at position P2 of a tetrapeptide analogue via solution-phase fragment condensation. The manufacturing process, executed in a 500 L glass-lined reactor with a retreat-blade impeller operating at 80–100 rpm, begins with the pre-activation of the Boc-protected acid (0.95 kg, 1.0 equivalent) using ethyl chloroformate (1.05 equivalents) and N-methylmorpholine (1.10 equivalents) in anhydrous THF at −15 ± 3°C for 20 minutes to form the mixed anhydride. A batch-recorded deviation from this temperature window—specifically, excursions above −10°C—resulted in a 2.3% yield reduction attributed to urethane disproportionation, as confirmed by FTIR monitoring of the carbonyl stretch at 1825 cm⁻¹ shifting to 1800 cm⁻¹. The mixed anhydride is subsequently quenched with the H-Pro-OtBu ester free base in a single-port addition over 15–20 minutes, maintaining internal temperature below −5°C, to assemble the P2-P3 dipeptide fragment. It is critical that the methoxymethyl substituent remains intact throughout this sequence; the ether is stable to the mixed anhydride conditions but undergoes ~8% cleavage if the quench pH during aqueous workup exceeds 9.5, as evidenced by LC-MS detection of the corresponding 4-hydroxymethyl degradation product (M+H⁺ = m/z 216.1). The dipeptide is purified by silica gel plug filtration (eluent: ethyl acetate/heptane 1:1 v/v) and advanced to Boc deprotection, which is accomplished with methanolic HCl generated in situ from acetyl chloride (2.5 equivalents) in methanol at 0°C to room temperature over 4 hours. The final active pharmaceutical ingredient candidate, an N-acylprolylpyrrolidine bearing a nitrile warhead, emerges from subsequent coupling with a P1 fragment and is classified as a prolidase-stable POP inhibitor with nanomolar potency (exact Ki values for specific structural analogs are reported in peer-reviewed medicinal chemistry literature). Analytical release criteria for the dipeptide intermediate mandate chiral purity analysis per EP monograph 2.2.28 (capillary electrophoresis using a neutral coated capillary with a sulfobutyl ether-β-cyclodextrin background electrolyte at pH 2.5), with acceptance threshold set at <0.15% of the (2R,4R)-enantiomer. Storage of the Boc-protected building block itself requires desiccated conditions (<30% relative humidity) at 2–8°C; accelerated stability studies at 40°C/75% RH over 6 months documented 0.7% total related substances increase, confirming adequate stability for ambient shipment with cold packs per WHO/GMP Annex 9 guidelines for time- and temperature-sensitive pharmaceutical starting materials.

Inhibiting the Exosite: 4-Methoxymethylproline as a Scaffold for Allosteric Modulation of Coagulation Factor XIa

A parallel discovery program identified (2S,4S)-1-(tert-butoxycarbonyl)-4-(methoxymethyl)pyrrolidine-2-carboxylic acid as a key intermediate for a series of non-competitive Factor XIa (FXIa) allosteric inhibitors targeting the interface between the catalytic domain and the apple A3 domain. The pyrrolidine ring bearing the methoxymethyl side chain serves as a rigidified analogue of a leucine-like moiety that occupies a cryptic hydrophobic subpocket (residues Tyr143, Leu144, Ile151, Lys192) revealed only upon active site ligation, a feature that necessitates an orthogonal screening paradigm using a two-step Surface Plasmon Resonance (SPR) assay on Biacore 8K instrumentation with FXIa immobilized at 8000–10000 RU on a CM5 chip at 25°C. The incorporation ratio for building block derivatization via amide coupling at the C-terminus employs HOBt/DIC activation in dichloromethane at 0°C with a stoichiometry of 1.0:1.2:1.2:1.15 (acid:amine:HOBt:DIC), producing a P2-P1′ fragment that is further elongated at the N-terminus following Boc removal with TFA/CH₂Cl₂ (1:1 v/v) containing 2% triisopropylsilane as a cation scavenger. During kilo-scale manufacture of the final drug substance intermediate, continuous flow chemistry (Corning Advanced-Flow G1 reactor, 2.0 mL internal volume per plate, 8 plates in series) replaced the batch deprotection step to mitigate exotherm risks, achieving a residence time of 45 seconds at 25°C with 99.8% conversion. The downstream FXIa inhibitor molecule produced—a class of small-molecule anticoagulants distinguished by minimal bleeding risk in tail-transection models at supratherapeutic doses (pharmacology data cross-referenced in peer-reviewed hematology journals)—is formulated as an oral prodrug via phosphate ester prodrug strategy at the P1′ phenol. Regulatory compliance is evaluated against ICH S7A (cardiovascular safety pharmacology) and ICH S7B (hERG assay) guidelines; the allosteric character of the target interaction is confirmed by demonstrating saturable shift of the IC₅₀ value for a tripeptide substrate (S-2366) cleavage at FXIa concentrations ranging from 0.5 nM to 50 nM, an assay configuration explicitly prescribed in FDA guidance for non-active-site anticoagulant characterisation. The required personal protective equipment during synthesis includes nitrile gloves with breakthrough time above 240 minutes for dichloromethane and full-face supplied-air respirators during the TFA deprotection step, per the process safety assessment document filed with the authorized contract manufacturing organization.

Is a Boc-Protected Pyrrolidine Core Advantageous in the Synthesis of Pan-Genotypic NS5B Polymerase Thumb Pocket II Inhibitors?

Within the chemical space of palm-adjacent thumb pocket II (TP-II) non-nucleoside inhibitors of hepatitis C virus RNA-dependent RNA polymerase (NS5B, genotype 1b HCV Con1 replicon), the lipophilicity and conformational restriction imparted by the 4-methoxymethyl substituent of (2S,4S)-1-(tert-butoxycarbonyl)-4-(methoxymethyl)pyrrolidine-2-carboxylic acid contribute to a measurable enhancement in replicon potency relative to the corresponding 4-unsubstituted proline congener. The building block is incorporated at position R3 of the prototypical benzimidazole-indole carboxamide scaffold via an HATU-mediated amide coupling with 6-amino-2-(4-chlorophenyl)-1H-benzimidazole at 0.85 equivalents of the aminoindole (adjusted due to the competitive acylation of the benzimidazole N1 position, which occurs at rates ~2x lower for the sterically shielded Boc-protected acid versus unsubstituted Boc-proline as determined by competitive reaction calorimetry data published in process chemistry journals). The coupling is carried out with 1.5 equivalents of DIPEA in DMF at 0°C to room temperature over 16 hours under a nitrogen headspace. Post-acylation Boc removal with 4.0 M HCl in 1,4-dioxane at 10–15°C generates the more water-soluble hydrochloride salt, which undergoes final sulfonylation with methanesulfonyl chloride (1.2 equivalents) in pyridine at 0°C to install the critical P2 sulfonamide moiety hypothesized to engage a hydrogen-bond network with NS5B residues Lys445, His446, and Cys366. Purification via flash chromatography (normal-phase SiO₂, gradient elution from neat dichloromethane to 5% methanol in dichloromethane) yields the free base of the target benzimidazole-amide, which is subsequently crystallized from ethyl acetate/hexane (1:3 v/v) to provide material with a melting point of 168–170°C and a purity exceeding 99.0% by HPLC area. The final marketed drug product class encompasses single-tablet regimen components administered with a nucleotide-based NS5B inhibitor and an NS5A inhibitor, as reflected in combination therapy guidelines published by AASLD/IDSA. The supply chain for this intermediate is regulated under the FDA’s Quality by Design (QbD) paradigm as elaborated in ICH Q8(R2); a design space verification study establishing the permissible range for the methoxymethyl ether oxygen content—<0.05% 4-(methoxymethyl)prolinol as a process-related impurity—must conform to ICH Q11 principles for the selection and justification of starting materials for synthetic drug substances.

Formulations of lipid nanoparticle (LNP)-encapsulated siRNA constructs targeting hepatocyte-expressed SERPINA1 mRNA for the treatment of alpha-1 antitrypsin deficiency employ a GalNAc-conjugated delivery agent that is anchored to a cholesterol-derived spacer using a polyproline type II (PPII) helix-inducing peptoid linker. (2S,4S)-1-(tert-Butoxycarbonyl)-4-(methoxymethyl)pyrrolidine-2-carboxylic acid serves as a monomer in the solid-phase assembly of this oligoproline linker at a loading ratio of 0.5 mmol/g on ChemMatrix Wang resin functionalized with a photolabile 4,4′-dimethoxybenzhydryl linker. The synthesis protocol on a Liberty Blue automated microwave peptide synthesizer (CEM Corporation) programs double-coupling at 90°C for 110 seconds with 25 W microwave power using 4.0 equivalents of the building block and 3.9 equivalents of Oxyma Pure/DIC in DMF; single-coupling protocols yielded a deletion peptide content exceeding 12% as measured by LC-MS, attributed to the steric shielding of the secondary amine by the bulky methoxymethyl substituent which retards acylation kinetics. Following the assembly of 6 consecutive 4-methoxymethylproline units (producing a rigid rod spacer of approximately 18–20 Å in length by molecular dynamics simulation in explicit SPC/E water), the N-terminal Fmoc group is cleaved with 20% piperidine in DMF and the GalNAc triantennary cluster is installed using a pre-formed pentafluorophenyl ester. The entire construct is cleaved from the resin by UV irradiation at 365 nm (100 W mercury lamp, 2 hours, 0°C in methanol) to preserve the Boc protecting group for subsequent formulation steps downstream. The terminal product is the LNP-formulated GalNAc-siRNA conjugate, which accumulates in hepatocytes via ASGPR-mediated endocytosis; biodistribution data confirming this mechanism is available in toxicokinetic reports filed under IND applications for rare-disease siRNA modalities. The manufacturing process for the linker fragment must adhere to ICH Q3C guidelines defining residual solvent limits for DMF (Class 2, PDE 8.8 mg/day) and piperidine (not classifiable, but controlled in the final siRNA drug substance specification to <3.0 ppm by headspace GC-MS). Any batches intended for use in a Phase II clinical supply chain require lyophilized storage at −20 ± 5°C under argon in Type I borosilicate glass vials sealed with fluoropolymer-coated stoppers to avoid the hydrolytic ring-opening of the pyrrolidine amide bonds observed at 4°C in unbuffered aqueous solution above pH 6.0 over 4 weeks storage (forced degradation data reported in pharmaceutical development sections of common technical documents).