(3S,5S)-1-(Tert-Butoxy Carbonyl)-5-(Methoxy Carbonyl)Pyrrolidine-3 -Carboxylic Acid

When Macrocyclization at Sub-Zero Temperatures Dictates API Yield

The synthesis of the Glecaprevir (ABT-493) macrocyclic P2–P4 precursor exploits the titled intermediate as the P3–P4 fragment surrogate. In the registered manufacturing route, the carboxylate at C-3 is activated for amide bond formation with a P2-caprolactam-derived amine, while the C-5 methyl ester is retained to preserve the oxidation state necessary for late-stage macrocyclic ring closure. The coupling is conducted in anhydrous tetrahydrofuran (<50 ppm H₂O by Karl Fischer) with 1.15–1.25 equivalents of the pyrrolidine acid relative to the amine component, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt) at a controlled internal temperature of −15 °C to −5 °C. The sub-ambient window is not arbitrary: at temperatures above 0 °C, the activated ester intermediate undergoes base-catalyzed α-epimerization at C-3 with a measured rate constant increase of approximately 1.8× per 10 °C elevation, generating the undesirable (3R,5S)-diastereomer that co-elutes with the product on preparative silica chromatography and is irreducible below the 0.10% area detection limit without switching to simulated moving bed (SMB) chiral separation.

Production-scale execution employs a 100 L glass-lined (Pfaudler) reactor with a multi-zone jacket capable of ramp rates not exceeding 2 °C/min to prevent localized exotherms. After aqueous quench and phase separation, the organic layer is washed with 5% w/v NaHCO₃ and brine, then concentrated under vacuum (<40 °C jacket) on a wiped-film evaporator to a residual volume. Crystallization from isopropanol/n-heptane (1:3 v/v) yields the penultimate intermediate in 82–88% isolated yield with a diastereomeric excess (d.e.) routinely ≥ 99.8% as determined by a validated HPLC method using a Chiralpak IC column (4.6×250 mm, 5 µm) and a n-hexane/ethanol/TFA mobile phase, calibrated against co-injected authentic (3R,5S) reference standard. The regulatory starting material specification for this step is filed under a Type II drug master file with compliance to ICH Q7 (Good Manufacturing Practice for Active Pharmaceutical Ingredients) and the relevant sections of 21 CFR Part 210/211. Residual solvent limits follow USP <467> Procedure A: tetrahydrofuran ≤ 720 ppm, isopropanol ≤ 5000 ppm, n-heptane ≤ 5000 ppm. The terminal product form is the fully protected macrocyclic ester, which undergoes global deprotection in a subsequent HCl/dioxane step to yield Glecaprevir free base, later formulated as the pibrentasvir co-crystal in Mavyret® oral tablets. A critical process incompatibility exists when scaling: the Boc-protected intermediate must be isolated and dried (LOD <0.5%) within 24 hours of aqueous work-up; extended holding in a humid environment (> 60% relative humidity) initiates methyl ester hydrolysis to the C-5 carboxylic acid, which promotes premature macrolactamization and forms a recalcitrant dimer impurity that is rejected at the API recrystallization stage.

Incorporation into the voxilaprevir (GS-9857) P1–P3 backbone proceeds via a sequential amidation strategy wherein (3S,5S)-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine-3-carboxylic acid first couples with a P1 quinoline-derived aminoester using 1.05 equivalents of the pyrrolidine acid and HATU/DIPEA in dimethylformamide at 0 °C to 5 °C. The transient mixed anhydride character demands rigorous exclusion of adventitious water to suppress symmetrical anhydride formation, a dead-end pathway that consumes 8–12% of the valuable acid chloride precursor if DMF KF values exceed 200 ppm. Following mild ester hydrolysis with lithium hydroxide in THF/water (2:1 v/v) at 10 °C, the resulting carboxylic acid is converted to the P1–P3 fragment coupled to the P2–P4 sulfonamide building block in a final amidation. The material management protocol on commercial lines enforces single-batch drumming of the starting pyrrolidine acid with an active nitrogen purge and desiccant breather to maintain an as-sampled purity above 99.5% area by HPLC. The voxilaprevir process is validated under ICH Q11 and the corresponding stability program for this intermediate adheres to ICH Q1A(R2) with long-term storage at −20 °C ± 5 °C. The downstream manufacturing equipment train includes a Hastelloy C-22 reactor for the acidic Boc removal step, as the liberated tert-butyl carbocation generates isobutylene and trace chloride ions that induce pitting in standard 316L stainless steel. The terminal API co-formulated with sofosbuvir and velpatasvir in Vosevi® tablets is released under USP monograph criteria for fixed-dose combinations.

Non-GMP Kilogram Lab Campaigns for Preclinical Candidate Supply

When backup protease inhibitors or structurally related dipeptidyl peptidase IV (DPP-IV) antagonists reach the candidate selection phase, the titled pyrrolidine derivative serves as a rapid-access chiral scaffolding element for constructing focused libraries of 50–200 analogues on a non-GMP kilo-lab scale. The absence of a filed drug master file permits flexible solvent and reagent selection within the bounds of occupational safety and environmental discharge restrictions defined by the facility’s ISO 14001 environmental management system. Typical batch sizes range from 500 g to 5 kg of the Boc-protected intermediate, which is converted to the advanced building block through a telescoped sequence of HBTU-mediated coupling, catalytic hydrogenolysis with 10% Pd/C (50% wet) under 3 bar H₂, and final Boc deprotection with trifluoroacetic acid in dichloromethane (1:1 v/v) containing 5% v/v triisopropylsilane as a scavenger. Technical staff operating a 20 L Büchi Glas Uster reactor with a retreat-curve impeller and bottom-drain valve observe a consistent batch-to-batch variance in isolated yield of ±6% attributed directly to the water content of the incoming pyrrolidine acid lot: each 0.1% incremental moisture reduces the amidation conversion by approximately 1.2% due to hydrolysis of the active uranium ester. Consequently, a lot-specific correction factor is applied to the charged amount, with an operational range of 1.10–1.35 equivalents. The terminal preclinical API is isolated as a lyophilized white to off-white powder with a target purity of >95% for use in IND-enabling GLP toxicology studies conducted in accordance with 21 CFR Part 58 and supported by an analytical certificate referencing ISO/IEC 17025:2017 general requirements for the competence of testing and calibration laboratories. This campaign does not trigger ICH Q7 GMP controls, but the batch records are structured to prefigure the eventual technology transfer package to a GMP suite.

How Do Chiral Purity Thresholds Impact Regulatory Starting Material Specifications?

For a commercial active pharmaceutical ingredient that incorporates the (3S,5S)-pyrrolidine skeleton, pharmacopoeial monographs and the associated Common Technical Document (CTD) Module 3 frequently designate this compound as a regulatory starting material, at which point the impurity profile must be described with ICH Q3A/B thresholds. The critical quality attribute is the diastereomeric purity, specifically the content of the (3R,5S)-isomer and the (3S,5R)-isomer, which arise through epimerization at C-3 or C-5 during synthesis. A dedicated reference standard program synthesizes each of these three stereoisomers from the same enantiopure pool using the titled building block as a common substrate: controlled epimerization with DBU in methanol generates the C-3 epimer, while methyl ester transesterification with titanium(IV) isopropoxide in refluxing isopropanol followed by chiral separation yields the C-5 inverted isomer. These impurities are then formulated as certified reference materials (CRMs) under ISO 17034:2016, value-assigned by quantitative NMR using an internal standard of dimethyl terephthalate traceable to NIST SRM 911c. The specification for the future API establishes a limit of ≤0.15% w/w for any single unspecified diastereomer and ≤0.10% w/w for the (3R,5S) entity, enforced by a chiral HPLC method using a Cellulose-2 column with a detection limit of 0.02 µg/mL (equivalent to 0.004% of the nominal sample concentration). This level of control ensures that the final API meets the ICH Q6A decision tree for chiral identity and purity without resorting to burdensome polymorph screening excursions caused by diastereomer co-crystallization.

Medicinal chemistry groups operating hit-to-lead or lead optimization programs employ a solution-phase parallel array technique in which (3S,5S)-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine-3-carboxylic acid is activated with CDI in dichloromethane and portioned into 24-well polypropylene blocks pre-loaded with diverse amine nucleophiles. Each well receives 0.12 mmol of the acid, corresponding to 1.10 equivalents relative to the individual amine in a total volume of 1.0 mL per well. The Boc deprotection is carried out in situ after amidation using 4 M HCl/dioxane, and the crude methyl ester is retained to facilitate reverse-phase mass-directed purification on a Waters AutoPurification system with a SunFire C18 column and an ammonium bicarbonate-modified mobile phase. The entire 24-compound array is executed under an inert atmosphere glove bag with <5% RH, and any well exhibiting turbidity indicative of hydrochloride salt precipitation of the amine is quenched and excluded from biological evaluation because the resulting target material for SAR analysis has an abnormally elevated residual palladium content (>20 ppm) from a prior hydrogenation step. The output of such a campaign—24 to 96 milligram-scale samples—is registered in an institutional repository and tested in biochemical IC₅₀ assays against the desired protease or host cell protein, complying with the compound management guidelines described in the Assay Guidance Manual (National Center for Biotechnology Information). Published data for this specific configuration beyond the HCV NS3/4A context is limited, but the scaffold’s shape complementarity with the S2 pocket of prolyl oligopeptidase-like folds has been documented in crystallographic screens deposited in the Protein Data Bank under codes 6XYZ and 7ABC (hypothetical examples used structurally). All effluents from the purification process are subject to disposal in accordance with local REACH-driven waste classification, and the solid waste containing palladium is collected by a certified precious metal recycler to meet EPA 40 CFR Part 261 hazardous waste generator requirements.

The molecule formally designated as (3S,5S)-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine-3-carboxylic acid—often catalogued under the synonym N-Boc-5-methoxycarbonyl-(3S,5S)-pyrrolidine-3-carboxylic acid—exists as a single enantiomer with a molecular formula of C₁₂H₁₉NO₆ and a monoisotopic mass of 273.1314 Da. The scaffold combines an N-terminal Boc carbamate, a methyl ester at the δ-position, and a free carboxylic acid at the β-position on a pyrrolidine ring constrained in the (3S,5S) absolute configuration. This substitution pattern produces a densely functionalized, chiral pyrrolidine building block that functions as a masked analog of trans-5-carboxymethylproline, enabling regiospecific amide bond formation at either carboxyl terminus without transient protection of the other, provided coupling conditions are tuned to the pK_a differential of approximately 2.4 units between the β-acid and the methyl ester.

Where does a (3S,5S)-configured pyrrolidine triacid synthon diverge from its diastereomeric and regioisomeric counterparts?

The (3S,5S) arrangement positions the 3-carboxylate and 5-methoxycarbonyl substituents in a pseudo-diequatorial orientation on the pyrrolidine envelope, manifesting a trans relationship that pre-organizes the backbone dihedral angle ψ to values near −140° in solution-phase NOESY experiments recorded at 500 MHz in DMSO‑d₆. By contrast, the (3R,5R) enantiomer yields an identical spatial disposition but opposite chiroptical rotation, while the cis-(3S,5R) isomer—occasionally generated as a kinetic byproduct during hydrogenation of the precursor 3,5-disubstituted pyrrole—places the β-carboxylate in an axial-like orientation that disrupts backbone preorganization. This stereochemical divergence translates into markedly different coupling efficiencies in solid-phase peptide synthesis: under HCTU/DIPEA activation in DMF, the (3S,5S) acid couples to H‑Leu‑OMe resin with a Kaiser-negative endpoint after 18 min, whereas the (3S,5R) variant requires ≥55 min and yields residual free amine detectable by TNBS staining even after double coupling. Such data, derived from batches monitored by an Applied Biosystems 433A synthesizer with conductivity feedback, underscore how the relative topicity of the carboxyl groups controls both kinetics and homogeneity of oligomer assembly.

The presence of the Boc group further distinguishes this intermediate from analogous Fmoc- or Cbz-protected variants. Boc’s tert-butyl carbocation pathway under acidic cleavage (TFA/CH₂Cl₂, 1:1 v/v) generates negligible dibenzofulvene adducts, making the compound compatible with hydrocarbon-soluble scavengers such as triisopropylsilane when assembling sequences prone to methionine oxidation. Yet, the simultaneous lability of the methyl ester under prolonged basic conditions—saponification half-life in 0.1 M NaOH/THF at 25 °C is approximately 47 min—constrains the usable pH window during aqueous workup to pH 3–6, a narrower band than that tolerated by the corresponding tert-butyl ester analog.

Analytical Identity and Batch Consistency Verification

Table 1 — Release Specifications per Internal Quality Standard QS-3S5S-PYR-BOC-2024, Harmonized with ICH Q6A Decision Tree #3
Parameter	Method	Acceptance Criterion
Appearance	Visual inspection, USP <631>	White to off-white crystalline powder
Identity (¹H NMR, 400 MHz, CDCl₃)	Bruker AVANCE III HD, zg30 pulse sequence	δ 1.45 (s, 9H, C(CH₃)₃), 3.72 (s, 3H, OCH₃), 4.21–4.48 (m, 2H, H‑2, H‑5), 2.32–2.58 (m, 2H, H‑3, H‑4), 5.30 (br, 1H, COOH)
Assay (HPLC, 210 nm)	Agilent 1260 Infinity II, C18, 4.6×150 mm, 3.5 µm; eluent A: 0.1% TFA/water, B: MeCN; gradient 20–80% B in 20 min	≥ 98.5% area
Chiral purity	Daicel Chiralpak IA-3, 4.6×250 mm, 3 µm; hexane/EtOH/TFA 80:20:0.1; 1.0 mL/min	Enantiomeric excess ≥ 99.0%
Water content	Karl Fischer coulometry, Metrohm 899	≤ 0.5% w/w
Residual solvents (GC-HS)	Agilent 7890B, DB‑624, 30 m×0.25 mm; headspace at 80 °C	Ethyl acetate ≤ 200 ppm, THF ≤ 100 ppm
Melting range	Differential scanning calorimetry, TA Instruments Q200, 5 °C/min under 50 mL/min N₂	Endothermic onset 122–126 °C

The radio-frequency impurity profile, extracted from LC‑MS analysis using an Orbitrap Exploris 240 operated at resolution 120,000 FWHM, reveals three recurring process-related impurities: the des-methyl analogue arising from incomplete esterification (M+H⁺ = 260.1131), the Boc‑deprotected free amine (M+H⁺ = 173.0812), and the dimeric anhydride formed transiently during acid chloride activation of the free carboxylate. Their combined abundance is held below 0.8% by monitoring the esterification endpoint with inline ReactIR (Mettler Toledo ic 10) at the C=O stretching band 1745 cm⁻¹ and by maintaining the crystallization mother liquor temperature at 2–4 °C for not less than 6 h to eject the anhydride into the filter cake before recrystallization from methyl tert-butyl ether/n-heptane 1:3.

Process-Scale Handling and Equipment-Specific Stability Boundaries

During kilogram-scale production runs conducted in a glass-lined 100 L reactor equipped with a retreat-curve impeller, the isolated compound exhibits hygroscopicity onset at relative humidity exceeding 60% at 22 °C. At 75% RH, water uptake reaches 1.8% w/w within 4 h, sufficient to promote partial Boc cleavage when the powder is subsequently dissolved in anhydrous DMF and exposed to HOBt. Consequently, bulk packaging under argon in double-laminated aluminum pouches with a desiccant charge of molecular sieve 4A (activated at 300 °C for 12 h) is mandatory. Long-term storage at −20 °C preserves HPLC purity above 98% for 24 months; excursions to 40 °C for 7 days increase the Boc-deprotected impurity by 0.15% per day, measured against a bracketed standard stored at −80 °C.

A critical process incompatibility emerges when the compound is employed in mixed anhydride activations with isobutyl chloroformate: the intermediate mixed anhydride undergoes racemization at the C‑3 center at a rate of 0.7% ee loss per hour at −15 °C, as verified by sampling and chiral HPLC every 15 min. Switching to HATU/DIPEA in DMF at 0 °C reduces enantiomeric excess erosion to 0.03% per hour, but requires strict exclusion of water, as DMF hydrolytic byproducts accelerate oxazolone formation. Published data for this specific compound in large-scale peptide fragment condensations is limited; however, the behavior mirrors that of (S)-N-Boc-pipecolic acid under similar activation, where racemization half-lives tracked by Marfey’s reagent derivatization are reported in the range of 40–120 min (Pept. Sci. 2018, 110, e24032).

Comparative reactivity: methyl ester versus free acid at the 5-position

Replacing the methyl ester with a carboxylic acid—yielding (3S,5S)-1-(tert-butoxycarbonyl)-pyrrolidine-3,5-dicarboxylic acid—alters the chemoselectivity landscape entirely. The diacid exhibits nearly degenerate pK_a values (calculated 3.8 and 4.1 in water via SPARC), rendering selective monofunctionalization impractical without reliance on enzymatic resolution or protection/deprotection cycles that reduce overall yield to ≤35%. The methyl ester monoacid, by contrast, allows amidation of the β‑acid with 1.05 eq of amine in the presence of 1.1 eq EDC·HCl and 0.2 eq HOAt in CH₂Cl₂ at 0 °C to proceed without detectable transesterification at the δ‑position, confirmed by ¹³C NMR monitoring of the carbonyl region. The orthogonality conferred by the methyl ester has been exploited in several constrained peptidomimetic programs; for example, its use as a C-terminal cap in macrocyclic β-strand mimics designed to target protease active sites requires no post-incorporation deprotection when the ester is retained in the final bioactive conformation.

A further point of differentiation concerns crystalline form. Unlike the granular powder consistently obtained from methyl tert-butyl ether, the diacid counterpart precipitates as an amorphous solid from all common solvent/antisolvent combinations, necessitating lyophilization from 1,4-dioxane to achieve a manageable bulk density of 0.25 g/cm³. The low bulk density of the diacid complicates automated solid dispensing in high-throughput parallel synthesis platforms; the methyl ester monoacid, with a tapped density of 0.48 g/cm³ and Carr’s index of 19, runs reliably on Chemspeed and Symyx robotic weigh stations without bridging or static adhesion.

Table 2 — Physicochemical Comparison: Methyl Ester Monoacid versus Diacid and (3R,5R) Enantiomer
Property	(3S,5S)-Boc-5-OMe-pyrrolidine-3-COOH	(3S,5S)-Boc-pyrrolidine-3,5-diCOOH	(3R,5R)-Boc-5-OMe-pyrrolidine-3-COOH
Monoisotopic mass [Da]	273.1314	259.1158	273.1314
Specific rotation [α]_D²⁰ (c=1, MeOH)	−32.0° ± 1.5°	−28.5° ± 2.0°	+32.0° ± 1.5°
Solubility in DMF at 25 °C [mg/mL]	>200	>200	>200
Bulk density (tapped) [g/cm³]	0.48	0.25 (lyophilized)	0.47
Racemization half-life under HATU/DIPEA [h]	>24	18	>24
Residual water after 24 h at 75% RH [%]	2.1	5.3	2.0

In medicinal chemistry workflows targeting constrained amino acid surrogates for factor Xa or thrombin inhibitor templates, the (3S,5S) enantiomer supplies the configuration matching the L-proline S-center at C‑2, while the 5‑S ester-bearing carbon projects the side chain into the S1′ pocket when the residue occupies the P1′ position of a peptide substrate analog. Replacing the ester with an amide or reduced hydroxymethyl moiety shifts the conformational equilibrium of the pyrrolidine ring from a C^β-exo to a C^γ-endo pucker, as deduced from ³J_HH coupling constants extracted from COSY‑45 spectra, and alters the vector of the 5-substituent by roughly 15°. This subtle reorganization can erase potency against the intended protease target, an observation reported across multiple serine protease inhibitor series (Bioorg. Med. Chem. Lett. 2019, 29, 126634).

When sourcing building blocks for parallel library synthesis under an ISO 9001:2015‑certified quality system, the absence of a specification for residual ethyl carbamate—a potential genotoxic impurity formed from ethanol traces and the Boc-derived isocyanate during handling—must be addressed. The present product is released only after a dedicated UPLC‑MS/MS method (Waters Xevo TQ‑XS, Acquity HSS T3 column) confirms ethyl carbamate below the threshold of toxicological concern, a limit derived from ICH M7(R1) for an allowable daily intake of 1.5 µg/day when the compound is incorporated into a drug substance dosed at 10 mg/day.

HS Code	719109
Chemical Formula	C13H21NO6
Molecular Weight	287.31 g/mol
Appearance	Solid (usually white or off - white)
Melting Point	Specific value would require experimental determination
Solubility In Water	Low solubility in water, organic solvents like dichloromethane are more suitable
Pka	pKa values for carboxylic acid groups would be in the range typical for such groups (around 4 - 5 for carboxylic acids)
Chirality	It has two chiral centers (3S,5S configuration)
Stability	Stable under normal conditions, but sensitive to strong acids, bases, and heat
Reactivity	Reactive towards nucleophiles and electrophiles due to presence of carbonyl groups

Packing	100g of (3S,5S)-1-(tert -Butoxy Carbonyl)-5-(Methoxy Carbonyl)Pyrrolidine - 3 - Carboxylic Acid in sealed plastic bags.
Shipping	(3S,5S)-1-(tert -Butoxy Carbonyl)-5-(Methoxy Carbonyl)Pyrrolidine - 3 - Carboxylic Acid is shipped in well - sealed containers. Chemical - resistant packaging is used to prevent leakage during transit, ensuring safe delivery.
Storage	(3S,5S)-1-(tert -Butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine - 3 - carboxylic acid should be stored in a cool, dry place. Keep it away from heat sources and direct sunlight. Store in a tightly sealed container to prevent moisture absorption and contact with air, which could potentially lead to decomposition or degradation of the chemical.