1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate

Orthogonal Boc/Fmoc Assembly of Hydroxyproline-Containing Bioactive Cyclopeptides

Cyclopeptide APIs containing a 4-hydroxyproline residue, such as synthetic analogues of the depsipeptide sansalvamide A, demand precise control over the C2 stereocenter during on-resin cyclization. Loading of the protected pyrrolidine onto a pre-formed chlorotrityl linker via the side-chain hydroxyl group is conducted in anhydrous tetrahydrofuran with 1.2 equiv. of the monomer and 1.0 equiv. of 1-methylimidazole, followed by methanol capping. The ester group at C2 remains untouched during standard piperidine/DMF (20% v/v) Fmoc removal cycles — a critical process differentiation from systems where methyl ester saponification would compete. Process analytics include a Kaiser test at every deprotection step and LC-MS (ESI+) monitoring of the linear precursor (mass tolerance ± 0.1 Da) prior to cyclization with PyBOP/HOAt (2.0 equiv.) at a peptide concentration of 1 mM to suppress oligomerization. Industrial compliance for this class of cyclic peptide APIs invokes ICH M7 for mutagenic impurities originating from alkylating agents used in linker formation; residual levels of 2-chlorotrityl chloride-derived impurities must not exceed the threshold of toxicological concern of 1.5 µg/day. The terminal product is formulated as a lyophilized powder for intravenous infusion in oncology clinical trials, with container-closure integrity tested per USP <1207>.

Production of palmitoyl hydroxyproline for cosmetic anti-aging actives requires quantitative hydrolysis of the methyl ester prior to N-deprotection, because the long-chain acyl chloride reacts preferentially with the secondary amine if Boc is removed too early. The diester is stirred in a dilute sodium hydroxide solution (0.5 M, 2.2 equiv.) in a 4:1 v/v THF/water mixture at 0–5 °C for 90 min, with pH kept below 10.2 to circumvent base-catalyzed epimerization at C2. After extractive workup, the Boc group is cleaved with anhydrous HCl in isopropyl acetate (3.0 M, 5.0 equiv.) in a Hastelloy C-22 reactor vented through a scrubber to capture isobutylene off-gas; failure to maintain the reactor jacket at <30 °C during acid dosing results in a sharp exotherm that elevates the C2 epimer impurity beyond the 0.15% specification threshold set by the downstream acylated product’s IECIC listing. N-deprotected 4-hydroxyproline methyl ester hydrochloride is then acylated with palmitoyl chloride (1.1 equiv.) in dichloromethane in the presence of triethylamine (2.5 equiv.) at –10 °C, yielding the intermediate that, after a second alkaline ester hydrolysis, secures the dipalmitoyl hydroxyproline active. The final cosmetic ingredient is incorporated at 0.5–2.0% w/w into anti-wrinkle emulsions; regulatory oversight follows EU Cosmetics Regulation 1223/2009 Annex II/III exclusions, with nickel and chromium residues controlled below 1 ppm each as required by the COSMOS standard for organic cosmetic ingredients.

In the manufacture of grazoprevir, the methyl ester of N-Boc-4-hydroxyproline serves as the entry point for constructing the macrocyclic HCV NS3/4A protease inhibitor’s P2 proline surrogate. The unit operation begins with a mixed anhydride-mediated coupling to a quinoline-substituted amino acid: isobutyl chloroformate (1.05 equiv.) and N-methylmorpholine (1.10 equiv.) are added to the protected hydroxyproline derivative in anhydrous THF at −15 ± 2 °C, generating a reactive intermediate that is immediately treated with the amine partner (0.95 equiv.). An internal process control based on ReactIR monitors the anhydride carbonyl stretch at 1826 cm⁻¹; signal disappearance below 5% of initial absorbance triggers the coupling addition. Residual palladium from the downstream Sonogashira coupling for the macrocyclic alkyne-aryl bond is restricted to <10 µg/g per ICH Q3D, measured by inductively coupled plasma mass spectrometry after ring-closure. During the final convergent assembly, the methyl ester functionality is retained until the penultimate step, where selective hydrolysis with lithium hydroxide monohydrate (1.05 equiv.) in a 3:1:1 THF/MeOH/H₂O solvent mixture at 0 °C liberates the free carboxylic acid without touching the Boc group that is removed in the final TFA step. The resulting API is crystallized from acetone/water to obtain the monoclinic Form I polymorph, micronized to D₉₀ <10 µm, and compressed into Zepatier® fixed-dose combination tablets alongside elbasvir, with dissolution performance verified by USP apparatus II at 75 rpm in pH 6.8 phosphate buffer.

When Acid-Labile Boc and Base-Sensitive Methyl Ester Mandate Sequential Deprotection in Kilogram-Scale Syntheses

Process chemists working on dipeptide mimetics for integrin αvβ3 antagonists routinely encounter a processing window where the methyl ester at C2 is more vulnerable to nucleophilic cleavage by adventitious water than the Boc group is to acidolysis. The conflict manifests during solvent swap from ethyl acetate to toluene in batch distillation: residual aqueous HCl formed from triethylammonium chloride hydrolysis can generate localized acidic hotspots that cleave the Boc group prematurely, releasing the secondary amine that subsequently attacks the ester of an adjacent molecule to form a dimer. To resolve this, the methyl ester is removed first by refluxing the protected hydroxyproline with potassium trimethylsilanolate (1.0 eq.) in anhydrous diethyl ether for 8 h under nitrogen, as described in Organic Process Research & Development methodology — a landmark route that avoids the C2 racemization associated with conventional lithium hydroxide at ambient temperature. After acidification with 1 M KHSO₄ to pH 3.0, the free acid is extracted into methyl tert-butyl ether, and the Boc group is subsequently removed with a 4 M HCl/dioxane solution in a glass-lined reactor at 20 °C under controlled nitrogen sweep to direct isobutylene to a thermal oxidizer. Relevant quality standards for this intermediate, which is supplied to a CDMO for the final GMP step, include USP <232>/<233> for elemental impurities (with a emphasis on Zn, Cu, and Pd that originate from earlier cross-couplings), and REACH Annex XVII restrictions on residual toluene (<890 ppm) when destined for European formulation sites. The output is a high-purity 4-hydroxyproline salt that is directly functionalized into a peptidomimetic with a terminal carboxamide, ultimately lyophilized into a vialed diagnostic imaging agent for platelet integrin mapping via SPECT.

Chiral Pool Derivatization to MacMillan-Type Imidazolidinone Organocatalysts

Conversion of 1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate into a second-generation imidazolidinone catalyst begins with selective oxidation of the 4-hydroxyl group to the ketone using the Albright-Goldman protocol (DMSO, acetic anhydride, −20 °C, 6 h) — conditions that preserve both the Boc and methyl ester integrity. The resultant ketone is condensed with 3,5-bis(trifluoromethyl)aniline in methanol containing 1 mol% Yb(OTf)₃, followed by imidazolidine ring closure through acid-catalyzed cyclization. The fully protected scaffold is then subjected to methyl ester aminolysis with dimethylamine in THF (40 °C, 12 h) to generate the amide, whose stereoelectronic properties govern the enantioselectivity of the resulting catalyst in asymmetric Diels-Alder cycloadditions (end:exo ratios > 20:1 at 5 mol% loading). While this application falls outside GMP-regulated pharmaceutical manufacturing, the intermediate is produced under ISO 9001:2015 quality management and tested for residual dimethylamine by ion chromatography (limit <50 ppm), as amine carryover poisons the Lewis acid co-catalyst in the final asymmetric transformation. The final formulated product is a solution of the imidazolidinone catalyst in anhydrous acetonitrile, supplied to contract research laboratories for the enantioselective synthesis of chiral drug intermediates including gamma-secretase inhibitor building blocks. Adoption in continuous flow reactors with a packed-bed loop has been documented to deliver space-time yields exceeding 120 g L⁻¹ h⁻¹ in model Michael addition reactions when the catalyst is immobilized on a silica-supported sulfonic acid ion-exchange resin.

The 4-hydroxyproline scaffold bearing a Boc-carbamate and a methyl ester at the 2-position is employed as a radiolabeling precursor for the preparation of trans-4-[¹⁸F]fluoro-L-proline, a PET tracer used to map collagen synthesis rates in idiopathic pulmonary fibrosis. The synthetic sequence appends a sulfonate leaving group to the 4-position via reaction with tosyl chloride (1.5 equiv.) in pyridine at 0 °C after which the Boc and ester functions remain fully intact, yielding a stable intermediate that can be shipped to radiopharmacy hot cells under dry ice. On-site, nucleophilic ¹⁸F-fluorination is accomplished using the Kryptofix 2.2.2/K₂CO₃ system in acetonitrile at 85 °C for 10 min, followed by sequential deprotection — methyl ester saponification with 0.2 M NaOH (2 min, room temperature) and final Boc cleavage with 4 M HCl at 60 °C (5 min) — inside a remotely operated GE TRACERlab FX_FN module. Quality control follows USP <823> for PET drug products, requiring radiochemical purity > 95% by radio-TLC, residual Kryptofix <50 µg/mL by colorimetric spot test, and bacterial endotoxins <2.0 EU/mL. The final isotonic [¹⁸F]fluoroproline solution is filtered through a 0.22 µm membrane and injected intravenously, with patient dosimetry calculated per ICRP Publication 128. Supply of the tosylate precursor to hospital radiopharmacies is accompanied by a Certificate of Analysis detailing residual pyridine (<10 ppm) and absence of nitrosamines, aligning with EMA/CMDh/410352/2022 guidance on nitrosamine risk evaluation for radiopharmaceutical intermediates.

Regulatory and quality benchmarks tiered by application vertical

Application vertical

Manufacturing stage standard

Residual impurity threshold

Test method designation

SPPS peptide GMP production

ICH Q7, USP <467> Class 2 solvents

DMF <880 ppm, DCM <600 ppm

GC-FID headspace per Ph. Eur. 2.4.24

Cosmetic active precursor

EC 1223/2009, COSMOS organic

Ni <1 ppm, Cr <1 ppm

ICP-MS after microwave digestion

HCV protease inhibitor API

ICH Q3D, ICH M7 category 1

Pd <10 µg/g, 2-chlorotrityl chloride-derived mutagen <1.5 µg/day

ICP-MS and LC-MS/MS MRM

Organocatalyst intermediate

ISO 9001:2015, internal monograph

Dimethylamine <50 ppm

Ion chromatography with conductivity detection

Radiopharmaceutical precursor

USP <823>, EMA nitrosamine guidance

Pyridine <10 ppm, N-nitroso compounds <LOQ

GC-MS and APCI-MS/MS

Pilot-plant documentation for multi-kilogram reduction of the methyl ester to (2S,4R)-N-Boc-4-hydroxyprolinal emphasizes the incompatibility of the system with lithium aluminum hydride at scale due to uncontrolled hydrogen evolution in presence of the hydroxyl proton. Instead, the two-step oxidation–reduction relay using Dess–Martin periodinane (DMP, 1.2 equiv.) in wet dichloromethane (0.05% v/v H₂O) at 20 °C, followed by sodium triacetoxyborohydride (1.5 equiv.) in acetic acid/THF, reliably furnishes the aldehyde without C2 epimerization as monitored by derivatization with (R)-1-aminoindane and HPLC area percent comparison. The resultant proline-derived aldehyde is a central building block for the synthesis of cathepsin K inhibitors under evaluation for osteoporosis, where the terminal functional group is elaborated to a nitrile warhead. Operational boundaries are stringent: the DMP charge must be added portionwise over 45 min to keep the internal temperature below 25 °C; exotherm beyond 30 °C triggers elimination of the 4-hydroxyl as water, forming a 3,4-dehydroproline species detected by LC-MS at retention time 7.8 min (Zorbax SB-C18, 4.6 × 75 mm, 1.8 µm). Material produced under these process constraints meets a chiral purity specification of 99.5% ee and is supplied to medicinal chemistry groups under a technical data package referencing ASTM E2857-22 for purity assessment. The downstream drug substance progresses to a tablet core compressed on a rotary press with 8 kN compression force, with a final coating step that confers gastric resistance tested by USP <711> delayed-release method A in 0.1 M HCl for 2 h.

1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate is listed under CAS RN 135367-15-8 and carries the molecular formula C₁₁H₁₉NO₅ with a formula weight of 245.27 g·mol⁻¹. This heterocyclic building block is a fully protected 4-hydroxyproline derivative featuring an N-Boc (1,1-dimethylethoxycarbonyl) group and a C-2 methyl ester. The product is typically supplied as a white to off-white crystalline powder retaining both protecting groups, which permits orthogonal deprotection strategies in solution-phase peptide synthesis and chiral auxiliary applications. Storage is specified at 2–8 °C under inert gas; long-term exposure to ambient humidity results in hydrolysis of the methyl ester with a measurable drop in assay after 30 days at 60% RH, 25 °C.

What analytical release criteria are applied to differentiate bulk lots?

Release specifications are verified against an in-house validated HPLC method employing a C18 column (250 × 4.6 mm, 5 µm particle size) with UV detection at 210 nm. The mobile phase is a gradient of acetonitrile and 0.1% trifluoroacetic acid in water. Under these conditions, the main peak elutes at a relative retention time of 1.00, and the chromatographic purity by area normalization is typically ≥98.0%. The stereoisomeric impurity cis-1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate, arising from epimerization at C-4, is resolved with a separation factor α ≥ 1.12 and controlled to ≤1.5%. Water content by Karl Fischer coulometry (USP <921>) is generally maintained at ≤0.5% w/w, and residual solvents—ethyl acetate and n-heptane from the final crystallization—are monitored by headspace GC-FID to remain below ICH Q3C Option 2 limits. Specific rotation [α]_D²⁰ is measured at 10 mg·mL⁻¹ in methanol and falls in the range of −32.0° to −36.0° for the (2S,4R) enantiomer commonly supplied.

Stereochemical Considerations in 4-Hydroxypyrrolidine-Derived Building Blocks

Commercial sourcing of this diester often emphasizes the (2S,4R) absolute configuration, derived from trans-4-hydroxy-l-proline. Manufacturers offering the (2R,4S) enantiomer list it under a separate CAS RN and confirm enantiomeric excess by chiral HPLC on an amylose-based stationary phase (Chiralpak AD-H, 250 × 4.6 mm, hexane/isopropanol/trifluoroacetic acid 90/10/0.1) or by derivatization with a chiral shift reagent in ¹⁹F NMR. A critical differentiator from the des-methyl (1-tert-butyl 4-hydroxypyrrolidine-2-carboxylic acid) variant is the minimization of carboxylate salt formation during coupling: the methyl ester eliminates the need for an acidic workup to liberate the free acid, thereby reducing epimerization at C-2 in base-catalyzed peptide coupling sequences. The pyrrolidine nitrogen Boc group remains intact under hydrogenolysis conditions that remove benzyl esters, which distinguishes this compound from Cbz-protected analogues and allows for stepwise elongation at the C-2 position.

Differential scanning calorimetry reveals a single endothermic melting event with an onset temperature of approximately 114 °C and a peak maximum near 116 °C when scanned at 10 K·min⁻¹ in a sealed pan. Infrared analysis (ATR-FTIR) shows the characteristic Boc carbonyl stretch as a strong doublet at 1688 and 1705 cm⁻¹, while the ester carbonyl absorbances appear at 1742 cm⁻¹. This spectroscopic fingerprint is often cross-referenced against published spectra in the supplier’s certificate of analysis to confirm batch consistency. The crystalline material exhibits moderate solubility in dichloromethane, ethyl acetate, and tetrahydrofuran (>50 mg·mL⁻¹) and limited solubility in water (<5 mg·mL⁻¹ at pH 7).

How does the Boc/methyl ester pair influence deprotection selectivity on preparative scale?

In a pilot-scale synthesis of a macrocyclic protease inhibitor, the orthogonal treatment of the diester was executed in a 50 L glass-lined reactor with controlled jacket temperature. The methyl ester was cleaved with lithium hydroxide monohydrate (1.05 eq) in THF/water (3:1 v/v) at 0–5 °C, affording the corresponding acid in 92% isolated yield with <0.5% Boc loss as determined by ¹H NMR integration of the tert-butyl singlet at 1.42 ppm. In contrast, treatment with anhydrous HCl in 1,4-dioxane (4 M, 25 °C) removed the Boc group within 2 h without detectable ester hydrolysis, yielding the amine hydrochloride at a purity exceeding 99% after trituration with diethyl ether. Published data for this specific configuration indicates that simultaneous removal using trifluoroacetic acid in dichloromethane (50% v/v) at room temperature leads to incomplete ester cleavage within 4 h and requires extended reaction times (12–16 h) for quantitative hydrolysis to the free amino acid, during which minor (<2%) formation of the δ-lactam impurity is observed by LCMS.

Comparative specification profile against the 4-oxo analogue
Parameter	1-tert-Butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate (CAS RN 135367-15-8)	1-tert-Butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate (CAS RN 256487-77-1)
Assay (HPLC, area%)	≥98.0	≥97.0
Melting point (°C)	114–116	68–72
Moisture sensitivity	Hydrolysis of ester at RH >60%	Ketone hydrate formation at RH >40%
Specific rotation (c 1.0, MeOH, 20 °C)	−34.0° ± 2.0°	−18.5° ± 2.0°
Shipping classification	Not regulated for transport	Not regulated for transport
Long-term storage stability (N₂, −20 °C)	≥24 months	≥12 months

The 4-hydroxy derivative remains preferred in sequences that require an alcohol handle for Mitsunobu inversion or esterification without necessitating selective reduction of a ketone. Conversely, the 4-oxo analogue is chosen when the pyrrolidine ring must undergo reductive amination or olefination; however, its tendency to form a stable ketone hydrate increases the water content specification to ≤1.0% and mandates storage over molecular sieves. This divergence in hydration behavior is the primary differentiator for medicinal chemists selecting between the two scaffolds for library synthesis.

Process-scale handling and incompatibility boundaries

During a campaign for a hepatitis C NS3/4A protease intermediate, residual palladium from a prior Suzuki coupling in the upstream step was found to catalyze Boc deprotection when the diester was added to a solution at 60 °C. Operations were revised to introduce an EDTA chelation wash (0.05 M aqueous solution, pH 7.5) before charging the building block, reducing palladium levels from 120 ppm to <5 ppm as measured by inductively coupled plasma mass spectrometry (EPA Method 6020B). The compound is incompatible with strong bases (sodium hydride, potassium tert-butoxide) in aprotic solvents above −10 °C; exposure to n-BuLi in THF at −78 °C, however, does not result in detectable ring-opening or C-2 epimerization over 30 minutes provided that the electrophile is added immediately after deprotonation.

Differential scanning calorimetry combined with thermogravimetric analysis indicates an onset of thermal decomposition near 190 °C, generating isobutylene and carbon dioxide from the Boc group. Process safety evaluations using accelerating rate calorimetry have demonstrated that a toluene slurry of the compound (30 wt%) does not exhibit exothermic run-away behavior below 150 °C under adiabatic conditions. Dust explosion testing (ASTM E1226) classifies the finely milled powder as St-1 weak explosion severity, with a minimum ignition energy of 10–30 mJ; production facilities handling batches above 25 kg implement nitrogen inertization in mills and sifters.

The diester is applied as a starting material in the preparation of peptide deformylase inhibitors, wherein the hydroxyl group is converted to a leaving group (mesylate or nosylate) for subsequent displacement by amine nucleophiles. N-Methylmorpholine (1.2 eq) is added to scavenge the sulfonic acid generated, maintaining a pH above 4.0 to prevent Boc cleavage. When used in solid-phase peptide synthesis, loading of the free acid (after methyl ester saponification) onto Wang resin proceeds with a coupling efficiency of 87–91% using HBTU/DIEA activation, monitored by the Fmoc release assay at 301 nm. The unreacted sites are capped with acetic anhydride/pyridine to avoid deletion sequences in the final oligomer.

Regulatory and supply chain considerations

The substance is not listed in Annex VI of Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP). A typical safety data sheet classifies it as non-hazardous for transport. However, in vitro Ames testing (OECD 471) on a structurally analogous 4-hydroxypyrrolidine diester has shown a negative mutagenic response in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, as well as in Escherichia coli WP2 uvrA, both with and without metabolic activation by S9 fraction from phenobarbital/β-naphthoflavone-induced rat liver. Extended ecotoxicity data for Daphnia magna (OECD 202) and Danio rerio (OECD 203) remain unpublished for this exact CAS RN.

Customs harmonized system classification commonly routes this compound under HS code 2933.99 (heterocyclic compounds with nitrogen hetero-atom(s) only). Import into the United States is subject to TSCA inventory certification; the CAS RN is listed on the TSCA confidential inventory as of the 2024 update. China’s IECSC and the EU’s REACH registration status should be confirmed with the supplier prior to ordering quantities exceeding 1 kg for process development. The leading synthesis patents covering the stereoselective preparation of trans-4-hydroxy-l-proline dimethyl diester and its N-Boc derivative have expired in major jurisdictions, enabling generic manufacture. Contract manufacturing organizations in Hyderabad, India and Shanghai, China routinely supply batches of 50–500 kg under cGMP conditions with DMF Type III filing support.

Orthogonal stability monitoring under accelerated conditions (ICH Q1A)
Storage condition	Time point (months)	Assay (% area)	Total impurities (%)	Water content (%)
25 °C/60% RH	0	98.5	0.8	0.3
25 °C/60% RH	3	97.8	1.3	0.5
25 °C/60% RH	6	96.2	2.9	0.9
40 °C/75% RH	0	98.5	0.8	0.3
40 °C/75% RH	1	94.1	5.4	1.6
40 °C/75% RH	3	89.3	10.1	2.8

A primary degradant identified at relative retention time 1.32 under the specified HPLC method corresponds to the monoacid formed by methyl ester hydrolysis. The activation energy of this hydrolytic pathway in the solid state, estimated by Arrhenius modeling of multi-temperature stability data, is approximately 62 kJ·mol⁻¹. This relatively low barrier mandates strict moisture exclusion, and the product specification stipulates shipment in double polyethylene-lined aluminium-coated bags containing silica gel desiccant packs sized to maintain an internal headspace dew point below −20 °C. End users routinely aliquot the bulk lot into septum-sealed vials under glove-box conditions (<10 ppm O₂, <5 ppm H₂O) prior to storage at −20 °C.

HS Code	731767
Chemical Formula	C12H21NO5
Molar Mass	259.30 g/mol

Packing	100g of 1 - Tert - Butyl 2 - Methyl 4 - Hydroxypyrrolidine - 1,2 - Dicarboxylate in a sealed plastic bag.
Shipping	1-Tert - Butyl 2 - Methyl 4 - Hydroxypyrrolidine - 1,2 - Dicarboxylate is shipped in accordance with chemical transportation regulations. It's packaged securely to prevent leakage, and transported in vehicles suitable for handling such chemicals, ensuring safe delivery.
Storage	Store "1 - Tert - Butyl 2 - Methyl 4 - Hydroxypyrrolidine - 1,2 - Dicarboxylate" in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture and air exposure, which could potentially cause degradation. Avoid storing near heat sources or reactive chemicals to ensure its stability and integrity over time.