1-Tert-Butyl 2-Methyl (2S,4R)-4-Hydroxypyrrolidine-1,2-Dicarboxylate
|
HS Code |
598390 |
| Chemical Formula | C12H21NO5 |
| Molecular Weight | 259.30 |
| Chirality | Contains chiral centers (2S,4R) |
As an accredited 1-Tert-Butyl 2-Methyl (2S,4R)-4-Hydroxypyrrolidine-1,2-Dicarboxylate factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of 1 - Tert - Butyl 2 - Methyl (2S,4R)-4 - Hydroxypyrrolidine - 1,2 - Dicarboxylate in sealed chemical - grade packaging. |
| Shipping | The chemical "1-Tert-Butyl 2-Methyl (2S,4R)-4-Hydroxypyrrolidine-1,2-Dicarboxylate" is shipped in specialized, leak - proof containers. It follows strict safety protocols for chemical transportation, ensuring secure transit to the destination. |
| Storage | Store “1-Tert -Butyl 2-Methyl (2S,4R)-4-Hydroxypyrrolidine-1,2-Dicarboxylate” in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and potential degradation. Store it separately from incompatible substances, and ensure the storage area is well - ventilated to avoid the build - up of any harmful vapors. |
In the synthesis of peptidomimetic inhibitors targeting the 3C-like cysteine protease (3CLpro) essential for viral replication of certain coronaviruses, the (2S,4R)-configured hydroxyproline derivative serves as a rigid, hydrogen-bond-donating P2 fragment. The intermediate is first subjected to N-Boc cleavage in anhydrous 4 M HCl/1,4-dioxane at 0–5 °C, with off-gas HCl neutralised through a caustic scrubber to avoid corrosion of 316L stainless steel reactor internals. After solvent displacement into dimethylformamide, the liberated secondary amine is coupled to a pre-activated indole-2-carboxylic acid derivative using 1.05–1.15 molar equivalents of the acid, 1.2 eq. of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.0 eq. of 1-hydroxybenzotriazole monohydrate at 18–22 °C for 16 h. Residual coupling agents are quenched with 0.5 M aqueous citric acid and washed with 5% sodium bicarbonate. The C2 methyl ester is subsequently hydrolysed with 1.2 eq. of lithium hydroxide in tetrahydrofuran/water (3:1 v/v) at 10 °C to a free carboxylic acid, which then enters a Dakin-West-type activation pathway en route to the electrophilic warhead—typically an aldehyde or α-ketoamide. Throughout this sequence, the diastereomeric purity of the C4 stereocenter is monitored by chiral HPLC on an CHIRALPAK AD-H column (250×4.6 mm, 5 µm) with hexane/ethanol/diethylamine 90:10:0.1 at 1.0 mL/min; acceptance criterion is ≤0.3 area% of the (2S,4S) epimer. Residual methanol and dichloromethane are controlled per ICH Q3C Option 1 limits of 3000 ppm and 600 ppm respectively, verified on a headspace GC-FID system calibrated against Class 2 solvent mixtures. The resulting terminal API falls into the category of 3CLpro inhibitors being developed under FDA Emergency Use Authorization or analogous veterinary regulatory pathways, where applicable VICH GL18 residual solvent compliance is demonstrated. The methyl ester intermediate is introduced at a corrected purity basis of ≥98.5% (HPLC, 210 nm), and its usage ratio precisely accounts for the batch-specific water content determined by Karl Fischer coulometric titration to avoid hydrolysis-driven yield losses exceeding 2.3%.What limits the processing window for acidolytic deprotection of the N-Boc group during dipeptidyl peptidase IV inhibitor assembly?When the hydroxyproline scaffold is incorporated into clinical candidates for the dipeptidyl peptidase IV (DPP-IV) enzyme class, the lability of the tert-butyloxycarbonyl protecting group under acidic conditions must be tightly coupled with the thermal sensitivity of the methyl ester and the C4 hydroxyl configuration. Deprotection with trifluoroacetic acid (30–50% v/v in dichloromethane) proceeds at 10–15 °C with a residence time not exceeding 45 min; exotherms beyond 22 °C trigger measurable transesterification at the C2 methyl ester when residual methanol from the quench partitions into the organic phase, generating the isopropyl ester impurity at levels up to 1.8% as confirmed by LC-MS on a Kinetex C18 column (100×3.0 mm, 2.6 µm). The subsequent amide coupling with a substituted β-alanine fragment employs 1.03–1.10 eq. of the deprotected hydroxyprolinate salt and 0.98 eq. of the acid chloride dissolved in isopropyl acetate, with triethylamine maintained at 1.5 eq. relative to the free amine. Process-scale production on a 500 L glass-lined reactor equipped with a dual-flight retreat-curve impeller requires jacket ramping at ≤0.5 °C/min during the acid chloride addition phase; deviations from this profile have been associated with racemisation at the C2 position exceeding the 0.15% threshold when measured against a certified reference standard of the (2R,4R) enantiomer. The target active pharmaceutical ingredient, a DPP-IV inhibitor aimed at improving β-cell function in type 2 diabetes, falls under ICH Q7 GMP guidance for active substances, and the finished dosage form must demonstrate dissolution per USP <711> Apparatus II at 50 rpm. Residual palladium levels—critical when a prior hydrogenolytic debenzylation step is used upstream—are quantified by inductively coupled plasma mass spectrometry and must remain below 10 µg/g to satisfy the EMEA/CHMP/SWP/4446/2000 guideline on metal catalysts. Addition levels of the 4-hydroxyproline intermediate relative to the β-alanine coupling partner are set at 1.05 eq. with a tolerance of ±0.02 eq., and the input batch is blended for 20 min under dry nitrogen to ensure homogeneity before weighing.Cathepsin K inhibitor backbone assembly via activated ester protocols—crystallisation-driven control of the C4 epimerCathepsin K inhibitors designed to limit bone resorption in postmenopausal osteoporosis frequently embed a (2S,4R)-4-hydroxyproline core as a conformationally constrained scaffold that directs the P1–P2 substituents into the S′ subsites of the enzyme. In this application, the methyl ester is retained as a protected carboxylate throughout the first six linear steps, then selectively hydrolysed at a controlled pH of 8.5 ± 0.2 using a phosphate-buffered pig liver esterase suspension (200 U/mmol substrate) at 37 °C to avoid deprotecting the N-Boc group. The downstream coupling to an aminothiazole fragment utilises 1.12 eq. of pentafluorophenyl ester of the hydrolysed intermediate, generated in situ with 1.0 eq. of pentafluorophenyl trifluoroacetate and 1.5 eq. of N-methylmorpholine. The crude amide is crystallised from ethyl acetate/n-heptane 1:3 (v/v) with a cooling rate of 0.3 °C/min from 55 to 5 °C; seeding with 0.1 wt% pure product at 38 °C is mandatory when the supernatant concentration of the (2S,4S) epimer exceeds 2.5 mg/mL, otherwise homogeneous nucleation enriches the undesired diastereomer in the crystal lattice by a factor of 1.7 observed through single-crystal X-ray diffraction studies on a Bruker D8 Venture diffractometer. The final active pharmaceutical ingredient belongs to a class of reversible covalent inhibitors whose release specification references USP <905> for content uniformity and ICH Q3D for elemental impurities, with chromium and copper limits set at 11 µg/day and 130 µg/day respectively for parenteral administration. The hydroxyproline intermediate enters the registered process at a batch scale equivalent to 85–110 kg of active cathepsin K inhibitor per campaign, with a required potency of ≥99.0% (anhydrous, solvent-free basis) and a site-specific manufacturing authorisation per EU GMP Part II.In solid-phase peptide syntheses targeting collagen-mimetic peptides or stapled α-helical macrocycles that require a hydroxylated proline residue at position i+3 or i+4, the N-Boc-protected methyl ester is chemically transformed into Fmoc-Hyp(tBu)-OH through a two-step sequence in a single vessel: selective methyl ester saponification with 1.05 eq. of lithium hydroxide in 4:1 methanol/water at 5 °C, followed by Boc-to-Fmoc exchange using 4.0 eq. of 9-fluorenylmethyl chloroformate and 10 eq. of sodium carbonate in dioxane/water. The resulting Fmoc derivative is loaded onto a pre-swollen Rink amide AM resin (0.47 mmol/g substitution) using 4.0 eq. relative to free amino sites with 3.85 eq. of HCTU and 8.0 eq. of N,N-diisopropylethylamine in dimethylformamide for 45 min at 70 °C on a CEM Liberty Blue automated microwave peptide synthesizer operating at 35 W magnetron power. Deprotection of the tBu ether protecting group is performed on-resin with 95% trifluoroacetic acid containing 2.5% triisopropylsilane and 2.5% water, with cleavage limited to 90 min at ambient temperature; prolonged exposure beyond 120 min leads to detectable O-acylation of the freed hydroxyl by the TFA-labile linker, reducing the final peptide yield by 4–7%. The finished peptide, comprising 12 to 28 residues with a C-terminal amide, is purified by preparative HPLC on a C18 column with a gradient of acetonitrile/water/trifluoroacetic acid and lyophilised to <0.5% moisture. Therapeutic peptide candidates in this pipeline are subject to ICH Q6B specifications for biotechnology-derived products, with host cell protein residuals determined by ELISA and aggregated species by size-exclusion HPLC on a TSKgel G3000SWxl column; the monomeric purity target is ≥98.0 area%. The starting hydroxyproline building block is charged into the Fmoc conversion step at a corrected weight accounting for a loss-on-drying value of ≤0.15% determined by thermogravimetric analysis at 105 °C.Phosphoramidite and diphosphine ligand syntheses reliant on the defined (4R) hydroxy configuration for asymmetric inductionChiral ligands deployed in rhodium- and iridium-catalysed asymmetric hydrogenation of enamide precursors to sitagliptin intermediates or dansyl-protected amino acids are constructed from the (2S,4R)-hydroxyproline scaffold by converting the C4 hydroxyl into a leaving group while preserving the pyrrolidine ring chirality. The intermediate is dissolved in dichloromethane and treated with 1.05 eq. of methanesulfonyl chloride and 1.2 eq. of triethylamine at −10 to −5 °C over 60 min; exotherms above 0 °C promote elimination to the Δ3,4-dehydroproline derivative, which must be limited to ≤0.5 area% by HPLC (210 nm). The mesylate is displaced with potassium diphenylphosphide (1.5 eq.) in tetrahydrofuran at −20 °C under argon, and the resulting diphenylphosphino-pyrrolidine is reduced with trichlorosilane in the presence of triethylamine to a secondary phosphine, subsequently coupled to a biphenyl backbone to generate a bidentate ligand. The ligand is used at a substrate-to-catalyst molar ratio (S/C) of 10 000:1 to 20 000:1 in hydrogenation of methyl (Z)-2-acetamidocinnamate at 0.3 MPa hydrogen pressure and 25 °C, achieving enantiomeric excesses above 96% for the (S)-product when the mesylation sequence has maintained a C4 ee of >99.5%. The phosphine product is purified by column chromatography on neutral alumina under nitrogen, and residual phosphine oxide is kept below 1.2 wt% as per 31P NMR quantification. The terminal catalyst is not a drug substance but a specialty fine chemical used under contract manufacturing arrangements governed by ISO 9001:2015 and OHSAS 18001; however, any residual pyrrolidine-derived intermediates in the ligand must comply with occupational exposure limits defined in the customer’s process safety data sheet, typically 50 µg/m³ for sensitising amines. Throughout the sequence, the addition ratio of the hydroxyproline mesylate to potassium diphenylphosphide must remain at exactly 1.0:1.50 because excess phosphide causes nucleophilic attack at the methyl ester, generating the corresponding amide and reducing ligand yield by 8–12%.A hydrolytically degradable nonwoven mesh for post-surgical adhesion prevention is produced by electrospinning a polymeric blend that contains poly[(2S,4R)-4-hydroxyproline methyl ester acrylate]-co-poly(ε-caprolactone) synthesised from the bifunctional pyrrolidine intermediate. The intermediate’s hydroxyl group is acrylated with 1.15 eq. of acryloyl chloride and 1.3 eq. of triethylamine in anhydrous tetrahydrofuran at 0 °C to afford the monoacrylate monomer, which is subsequently copolymerised with ε-caprolactone at a molar feed ratio of 30:70 using stannous octoate (0.1 mol% relative to total monomer) at 130 °C for 24 h in a vacuum-sealed Schlenk tube. The resulting amphiphilic copolymer exhibits a number-average molecular weight of 45 000–55 000 g/mol (GPC, polystyrene standards, THF) and is dissolved in 2,2,2-trifluoroethanol at 12% w/v for electrospinning onto a rotating mandrel collector at 15 kV and a tip-to-collector distance of 15 cm. The mesh is annealed at 42 °C under vacuum for 8 h to remove residual monomer below the ICH M7 threshold of toxicological concern (1.5 µg/day) as applicable for implantable devices. Cytotoxicity evaluation per ISO 10993-5 on L929 fibroblast cells requires a viability of ≥80% at 72 h extract incubation, and the degradation products are profiled by LC-MS to verify the absence of carcinogenic intermediates such as acrylamide. This class of absorbable medical device complies with ISO 13485:2016 for design and manufacture and requires sterilisation by ethylene oxide according to ISO 11135:2014, with residual ethylene oxide limits set at <4 mg/device per ISO 10993-7. The hydroxyproline-derived monomer is incorporated at 28–32 mol% into the copolymer chain, and its batch-to-batch stereochemical purity is confirmed by chiral gas chromatography on a Chirasil-Dex CB capillary column; deviations exceeding 0.8% of the (2S,4S) diastereomer alter the crystallinity of the caprolactone segments, shifting the in vitro degradation half-life from 14 days to 9 days at pH 7.4. |
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In solid-phase peptide synthesis, the introduction of a conformationally constrained proline surrogate often necessitates a building block that presents orthogonal protecting groups, a pre-installed stereogenic center at the 4-position, and the correct relative configuration to mimic the natural trans-hydroxyproline puckering observed in collagen triple helices. The compound designated 1-tert-butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (empirical formula C11H19NO5, molecular weight 245.27 g·mol−1) fulfills these criteria through a combination of an acid-labile Boc-type amino protection, a methyl ester that resists β-elimination under mild alkaline conditions, and a secondary alcohol positioned trans to the C-2 carboxylate. Its use circumvents the need for late-stage hydroxyl protection/deprotection sequences, provided that coupling reagents selected for amide bond formation do not promote O-acylation – a side reaction documented when using excess DIC/DMAP combinations without HOAt additive.
The stereochemical signature of the 4-hydroxyl group dictates the pyrrolidine ring pucker population, with 4R substitution favoring a Cγ-exo envelope conformation (pucker amplitude ~0.38 Å, phase angle ∼30° as determined by 1H–1H coupling constant analysis in D2O, 400 MHz). This pucker orients the hydroxyl substituent in a pseudo-equatorial position, aligning the H–Cβ–Cγ–H dihedral angle close to −160°, a geometry that is recognized by the active-site iron of 2-oxoglutarate-dependent dioxygenases. In contrast, the (2S,4S) epimer adopts a Cγ-endo pucker that forces the hydroxyl into a pseudo-axial orientation, altering the distance between the hydroxyl oxygen and the catalytic Fe(II) by approximately 0.7–0.9 Å. Published inhibition constants for human HIF prolyl hydroxylase-2 indicate that the (2S,4R) isomer exhibits an IC50 value approximately 10-fold lower than its (2S,4S) counterpart when the pyrrolidine ring is incorporated into a 2-oxoglutarate mimetic scaffold. This stereochemical dependence is not limited to enzymatic recognition; in small-molecule crystallography of the free amino ester hydrochloride, the (2S,4R) isomer crystallizes in the orthorhombic space group P212121 with one molecule per asymmetric unit, whereas the cis epimer tends to form solvated monoclinic crystals that exhibit greater lattice disorder and batch-to-batch variability in powder X-ray diffraction patterns.
When microwave irradiation is employed to accelerate amide coupling on a CEM Liberty Blue™ synthesizer, the thermal lability of the tert-butyl carbamate becomes the primary processing constraint. Differential scanning calorimetry of the neat solid shows an exothermic decomposition onset at 153°C, yet in DMF solution the half-life of the Boc group drops to 45 min at 80°C and to less than 8 min at 100°C, as monitored by inline UV absorbance at 254 nm. Consequently, microwave cycles programmed above 50°C with a power ceiling exceeding 30 W are known to trigger partial N-deprotection, leading to double insertion of the monomer and a characteristic +245 Da mass adduct in the crude product. The recommended SPPS protocol therefore limits each coupling step to 5 min at 50°C using 4 equiv of the building block, 3.9 equiv HATU, and 8 equiv DIPEA in DMF. In contrast, the Fmoc-protected analogue, 1-Fmoc-2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate, tolerates coupling temperatures up to 75°C under microwave conditions, but its use mandates piperidine-mediated deprotection, which is incompatible with formyl, acetate ester, or α-chloroacetyl groups that may be present in the resin-bound intermediate. The Boc/methyl ester pair thus becomes the preferred architecture when the target sequence contains base-sensitive side-chain protecting groups such as the 4-methoxytrityl (Mmt) group on cysteine or the 2-chlorotrityl linker itself.
The global market for chiral pyrrolidine scaffolds offers several regioisomeric and protection-group variants, each with distinct process compatibility windows. Table 1 summarizes the key handling and deprotection differences across four commercially available 4-hydroxyproline diesters bearing identical (2S,4R) stereochemistry but differing N- and O-protection strategies.
| Compound | N-Protecting Group | Ester | Deprotection Conditions | Critical Residue Risk | Application Limitation |
|---|---|---|---|---|---|
| 1-tert-Butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate | Boc | –OCH3 | TFA/TIS/H2O (95:2.5:2.5), 2 h, 25°C | Residual TFA (0.01% w/w) requires lyophilisation from 0.1 M HCl | Not suited for sequences requiring global acidolytic deprotection of Trt groups while retaining the pyrrolidine ring closure |
| 1-Fmoc-2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate | Fmoc | –OCH3 | 20% piperidine/DMF, 20 min, 25°C | Dibenzofulvene adduct removal requires RP-HPLC polishing | Incompatible with base-labile linkers (HMBA, oxime) |
| 1-Cbz-2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate | Cbz | –OCH3 | H2 (1 atm), 10% Pd/C, MeOH, 4 h | Residual Pd (≤5 ppm) exceeds ICH Q3D parenteral limit without scavenger treatment | Requires Pd-scavenging resin (Silicycle SiliaMetS Thiol) for API batches |
| 1-tert-Butyl 2-benzyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate | Boc | –OBn | H2 (1 atm), 10% Pd/C, EtOAc, 12 h | Residual Pd plus benzyl alcohol; ester hydrogenolysis competes with N-Boc cleavage at extended times | Used only where orthogonal ester removal is not required; lactonisation risk upon acid workup |
The analytical release panel for research-grade 1-tert-butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate relies on a multi-technique purity assessment aligned with ICH Q2(R1) guidelines for impurity profiling. Reverse-phase HPLC (column: Waters XBridge C18, 3.5 µm, 4.6 × 150 mm; mobile phase A: 0.1% TFA in water, B: 0.1% TFA in MeCN; gradient 10→90% B over 20 min; flow 1.0 mL·min−1; detection at 210 nm) routinely returns a main peak area >98.5% with the 2-epimer eluting at a relative retention time of 1.08 and the des-tert-butyl impurity (free amine) at 0.72. Chiral purity is determined by normal-phase HPLC on a Chiralpak AD-H column (250 × 4.6 mm, hexane/isopropanol 90:10, 1.0 mL·min−1) with the (2R,4S) enantiomer resolved at Rs ≥ 2.5; typical enantiomeric excess exceeds 99.5%. Water content by Karl Fischer coulometry (Metrohm 831 KF, oven method 140°C) is controlled below 0.5% because higher moisture accelerates methyl ester hydrolysis during storage. 1H NMR in DMSO-d6 (400 MHz) serves as identity confirmation: the C-2 methine proton appears as a doublet of doublets at δ 4.32 (J = 8.4, 4.1 Hz), the tert-butyl singlet integrates for 9H at δ 1.39, and the methyl ester resonates at δ 3.68. Residual solvents – typically ethyl acetate and n-hexane from crystallisation – are quantified by headspace GC-FID according to USP <467> Procedure A, with combined levels not exceeding 5000 ppm.
The secondary alcohol at C-4 is both a synthetic handle for late-stage diversification and a potential source of yield loss in sterically demanding couplings. In segment condensations where the pyrrolidine building block is activated as its HATU ester and reacted with a resin-bound pentapeptide possessing a hindered N-terminal valine, the unshielded hydroxyl competes for the activated carboxylate, forming an O-acyl isourea adduct that is slowly cleaved during the subsequent TFA global deprotection step. Quantitative HPLC-MS analysis of the cleavage cocktail reveals between 7% and 12% of the O-acylated by-product when coupling is performed at 0.1 M concentration in DMF without racemisation suppressants. Addition of 0.6 equiv HOAt relative to the amino acid derivative suppresses O-acylation to <2% by accelerating the formation of the active ester and minimising the lifetime of the mixed anhydride intermediate. The free hydroxyl can, however, be exploited for chemoselective introduction of phosphate, sulfate, or glycoside moieties prior to ring deprotection. Under Mitsunobu conditions (DIAD, PPh3, THF, 0°C to rt), the 4R-alcohol undergoes clean inversion to the 4S-azide, which can be reduced to the corresponding amine – a sequence used to access 4-aminopyrrolidine-based factor Xa inhibitors. The (2S,4R) compound thus differs fundamentally from the corresponding 4-deoxy analogue, 1-tert-butyl 2-methyl pyrrolidine-1,2-dicarboxylate, which is inert to such derivatisation and whose ring flexibility results in a broader distribution of backbone dihedral angles in the resulting peptide, often accompanied by a 0.5–1.5 log unit reduction in target binding affinity.
Long-term stability studies conducted under ICH Q1A accelerated conditions (40°C / 75% RH, sealed amber vials) indicate that the methyl ester hydrolyzes to the free acid at a rate of approximately 1.8% per month when residual water exceeds 0.8%, generating a polar impurity that elutes at the solvent front under reversed-phase conditions. Once the ring nitrogen is unprotected, the free 4-hydroxy acid is known to lactonize to a bicyclic oxazolidinone structure (observed m/z = 198.1 [M+H]+) upon exposure to mildly acidic conditions, including silica gel chromatography. For storage beyond 6 months, the bulk material is therefore subdivided into single-use, argon-flushed vials and maintained at −20°C; lyophilisation from dioxane rather than acetonitrile/water mixtures reduces residual moisture by an additional 0.2%. On a manufacturing scale, the compound is typically produced in batch sizes of 5–25 kg through an enzymatic resolution route: recombinant Alcalase® 2.4L selectively hydrolyzes the (2R,4S) enantiomer of the racemic N-Boc-4-hydroxypyrrolidine methyl ester, leaving the desired (2S,4R) diester untouched. The unresolved ester is then isolated by extraction and crystallized from methyl tert-butyl ether/n-heptane at −15°C to reach the enantiomeric excess target of ≥99.0%. Final purification employs a vacuum drying step (40°C, 10 mbar, 72 h) to bring the residual solvent profile within ICH Q3C Option 2 limits. This process avoids the palladium contamination inherent in hydrogenolytic Cbz or benzyl ester removal, making the Boc/methyl ester architecture the preferred starting material for preclinical candidates where total metals must be controlled below the 10 µg/day PDE threshold for parenteral administration.
