(3S,4S)-3-Amino-4-Hydroxypyrrolidine-1-Carboxylic Acid Tert-Butyl Ester
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HS Code |
613052 |
| Iupac Name | (3S,4S)-3-Amino-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester |
| Molecular Formula | C9H18N2O3 |
| Molecular Weight | 202.25 g/mol |
| Appearance | Typically a white to off - white solid |
| Melting Point | Specific value depends on purity, usually in a certain range |
| Solubility | Soluble in some organic solvents like dichloromethane, less soluble in water |
| Polarity | Moderately polar due to amino, hydroxyl and ester groups |
| Stability | Stable under normal conditions, but sensitive to strong acids and bases |
| Chirality | Has two chiral centers (3S,4S configuration), optically active |
As an accredited (3S,4S)-3-Amino-4-Hydroxypyrrolidine-1-Carboxylic Acid Tert-Butyl Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 grams of (3S,4S)-3 - Amino - 4 - Hydroxypyrrolidine - 1 - Carboxylic Acid Tert - Butyl Ester in sealed vial. |
| Shipping | (3S,4S)-3 - Amino - 4 - Hydroxypyrrolidine - 1 - Carboxylic Acid Tert - Butyl Ester is shipped in carefully sealed containers. Chemical - resistant packaging ensures safety during transit, following strict regulations for chemical shipments. |
| Storage | (3S,4S)-3 - Amino - 4 - Hydroxypyrrolidine - 1 - Carboxylic Acid Tert - Butyl Ester should be stored in a cool, dry place, away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and contamination. Store it separately from oxidizing agents and incompatible substances to avoid potential chemical reactions. |
The incorporation of a (3S,4S)-configured 1,2-amino alcohol motif directly into a peptide backbone as a conformationally restricted proline surrogate demands rigorous control over both the steric outcome and the integrity of the acid-labile Boc protection. On an automated Fmoc-SPPS platform operating with 0.1 mmol scale resin loading, pre-activation of the incoming Fmoc-amino acid is not required when the pyrrolidine scaffold is introduced via its free primary amine. The building block (1.5 equiv) is dissolved in anhydrous DMF containing 0.5 M DIPEA and coupled using HATU (1.45 equiv) over 45 min at 40 °C in a CEM Liberty Blue microwave reactor. Single-coupling efficiency, monitored by Kaiser test and quantitative Fmoc release at 301 nm, routinely exceeds 99.2% for the S,S diastereomer on Wang or Rink amide resins. Premature exposure to 20% piperidine during standard Fmoc deprotection raises minimal risk of Boc cleavage provided the cumulative contact time with base stays below 2 × 5 min. However, during global side-chain deprotection with Reagent K (82.5% TFA, 5% phenol, 5% H₂O, 5% thioanisole, 2.5% EDT), the Boc group is fully removed within 2.5 h; the liberated pyrrolidine nitrogen remains protonated and does not interfere with RP-HPLC purification on a 5 μm C18 column using a linear 5–45% acetonitrile gradient over 30 min. Trace racemisation at the C3 amine-bearing stereocentre is detectable only when coupling temperatures exceed 50 °C or when the carboxyl-activated species is the sterically hindered Boc-β-hydroxy acid itself, conditions typically avoided by using the amine as the nucleophile. Finished lyophilised peptidomimetics, intended as protease inhibitor leads, are released only when chiral HPLC confirms ≥99.5% de and LC-MS shows a single peak with the expected [M+H]⁺ mass within 0.1 Da of theory.How the Boc-Protected Amino Alcohol Prevents Late-Stage Epimerisation in Macrocyclic HCV Protease Inhibitor AssemblyIn the convergent synthesis of P2–P4 macrocyclic inhibitors targeting HCV NS3/4A, the integrity of the (3S,4S) configuration at the pyrrolidine ring junction is the dominant factor controlling nanomolar potency. When the building block is deployed as a P2 surrogate, its unprotected hydroxyl group is first silylated with TBDMSCl (1.2 equiv, imidazole, 0 °C) to prevent participation in undesired intramolecular acyl transfer during subsequent amide bond formation. The free amine reacts with an activated P3 carboxylic acid—most commonly a Boc- or Cbz-protected α-amino acid—using EDC·HCl and HOBt (1.05 equiv each) in DCM at −15 °C to suppress diketopiperazine formation. Residual water content in the reaction mixture must be maintained below 200 ppm (Karl Fischer) because water accelerates the generation of N-acylurea by-products that co-elute with the desired intermediate on flash chromatography. Once the linear tetrapeptide is assembled, the TBDMS group is cleaved with TBAF at 0 °C and the Boc protection of the pyrrolidine nitrogen remains intact, allowing orthogonal unmasking of the terminal acid for macrolactamisation. Ring closure via intramolecular HATU‑mediated coupling at 1 mM substrate concentration in DMF yields the macrocycle without affecting the Boc group; subsequent treatment with 4 N HCl in dioxane removes all acid-labile protection and furnishes the free amino‑alcohol. Process analytical technology (ReactIR 15) monitors the disappearance of the O–TBDMS stretch at 1250 cm⁻¹ and the lactam carbonyl shift from 1690 cm⁻¹ to 1650 cm⁻¹ to define precisely the end-of-reaction. Only batches with an HPLC purity (area percent at 210 nm) exceeding 99.0% and a specific rotation [α]D²⁵ of −18.3° ± 0.5° (c 1.0, MeOH) are passed forward to salt formation and spray drying, consistent with ICH Q7 guidelines for GMP intermediates.Chiral P,N-Ligand Precursor for Palladium-Catalysed Asymmetric Allylic AlkylationConversion of the vicinal amino-alcohol to an oxazoline-phosphine ligand framework exploits the rigidity of the pyrrolidine ring to set the absolute configuration of the metal-binding site. The hydroxyl group is first mesylated (MsCl, 1.2 equiv, Et₃N, DCM, 0 °C) and then displaced with a diaryl- or dialkylphosphine borane complex under SN2 conditions; inversion at the C4 centre is verified by X‑ray crystallography of the derived PdCl₂ complex. The free amine is condensed with an ortho-substituted benzoyl chloride to form the oxazoline under Mitsunobu-free conditions using 1.5 equiv of Burgess reagent in THF at 65 °C for 4 h. Retention of the Boc group on the pyrrolidine nitrogen prevents competing coordination to palladium during the subsequent metallation step and is removed only after complexation, using TFA–anisole (95:5 v/v) at 0 °C for 30 min. The resulting P,N-ligand produces enantiomeric excesses of 88–94% in the Pd-catalysed allylic alkylation of rac-(E)-1,3-diphenyl-2-propenyl acetate with dimethyl malonate (BINAP-matched conditions), as determined by CSP‑HPLC using a Chiralpak AD-H column. Ligand loading of 2.5 mol% and a substrate-to‑nucleophile ratio of 1:3 in CH₂Cl₂ at 25 °C achieve full conversion within 3 h. Crucially, the Boc-protected precursor exhibits shelf stability exceeding 24 months when stored under argon at −20 °C, whereas the deprotected amino-alcohol degrades within days due to air oxidation of the secondary amine. This long-term stability positions the derivative as the standard shipping form in ligand development kits distributed under inert atmosphere packaging complying with ASTM D4169-22 for distribution cycle integrity.A reproducible kilogram-scale route to a rigid bicyclic iminosugar for lysosomal glycosidase inhibition begins with the O‑alkylation of the unprotected hydroxyl group of the Boc-pyrrolidine scaffold. Deprotonation with NaH (60% dispersion, 1.1 equiv) in anhydrous THF at 0 °C generates the alkoxide, which is treated with 1.05 equiv of bromoacetaldehyde diethyl acetal and catalytic TBAI under reflux for 16 h. The acetal is hydrolysed in 1 M HCl at 50 °C to expose the aldehyde; reductive amination with the primary amine, simultaneously released upon Boc cleavage with TFA, triggers intramolecular cyclisation that delivers the fused [2.2.1]-azabicycloheptane core. The pH of the reductive amination mixture must be held between 4.5 and 5.0 using NaOAc buffer to prevent over-reduction of the imine intermediate, a condition monitored in-line with a Mettler Toledo InPro 3250 pH electrode. Crude iminosugar is purified over Amberlite CG‑50 (NH₄⁺ form) resin, eluting with 0.2 M aqueous ammonia. Tested against α‑L‑fucosidase from Thermotoga maritima according to a continuous fluorimetric assay (4‑methylumbelliferyl substrate, λex 365 nm, λem 450 nm), the derived hydrochloride salt exhibits a Ki of 0.47 ± 0.03 μM, competitive with the natural substrate. Full compliance with OECD 423 acute oral toxicity guidelines requires removal of free pyrrolidine by dialysis prior to submission; residual Boc‑protected precursor, however, is classified as a non-dangerous good for road transport (ADR) in its stable crystalline form, simplifying logistics from the CDMO to the biological testing facility.
All yields determined after RP‑HPLC purification and refer to diastereomerically pure product by chiral stationary phase analysis. Epimerisation quantified by comparison of peak area for (3R,4R) diastereomer versus total diastereomer area at 210 nm. Reaction monitored by LC‑MS on a Poroshell 120 EC‑C18 column, 2.7 μm, 4.6 × 50 mm.
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The building block (3S,4S)-3-Amino-4-Hydroxypyrrolidine-1-Carboxylic Acid Tert-Butyl Ester (also referred to as N-Boc-cis-3-amino-4-hydroxypyrrolidine) exhibits molecular formula C9H18N2O3 and a molecular weight of 202.25 g·mol⁻¹. The compound is isolated as an off-white crystalline powder with a melting range of 95–102 °C and a decomposition onset at 98 °C, as recorded by differential scanning calorimetry at 10 °C·min⁻¹ under nitrogen. The cis-1,2-amino alcohol geometry locks the pyrrolidine ring into a conformation that is exploited in peptidomimetic drug design, while the tert-butyl carbamate (Boc) protecting group renders the secondary amine compatible with a broad range of coupling and oxidation conditions. Commercial supply typically originates from batch syntheses conducted in 50 L glass-lined reactors under cGMP principles aligned with ICH Q7, yielding lots suitable for early-phase clinical candidate synthesis.
Each lot is released against a panel of analytical methods validated per ICH Q2(R1). The following specifications are applied to material destined for use as a registered starting material or advanced intermediate.
| Parameter | Specification | Method |
|---|---|---|
| Appearance | Off-white crystalline powder | Visual / Optical microscopy |
| Identification | Conforms to reference 1H and 13C NMR spectrum; IR spectrum matches reference | USP ⟨197⟩, ⟨854⟩, ⟨197K⟩; ATR-FTIR |
| Purity (HPLC, area %) | ≥ 99.0% | In-house method; C18, 210 nm; gradient MeCN/H2O 0.1% TFA |
| Any single impurity | ≤ 0.5% | Same HPLC method |
| Enantiomeric purity | ≥ 99.5% ee | Chiral HPLC; Chiralpak AD-H, 254 nm, hexane/EtOH/TFA |
| Specific rotation [α]D20 | −20° to −24° (c = 1.0, MeOH) | USP ⟨781⟩; Rudolph Autopol IV polarimeter |
| Water content (Karl Fischer) | ≤ 0.5% | USP ⟨921⟩ Method Ia |
| Residual solvents | Ethanol ≤ 5000 ppm; dichloromethane ≤ 600 ppm; all others conform to ICH Q3C Class 2/3 limits | GC-FID headspace per USP ⟨467⟩ |
| Heavy metals | ≤ 10 ppm | USP ⟨231⟩ Method II |
| Residue on ignition | ≤ 0.1% | USP ⟨281⟩ |
Crystallisation from heptane–ethyl acetate mixtures yields a Form I polymorph that exhibits consistent solubility in dichloromethane (>200 mg·mL⁻¹), tetrahydrofuran, and N,N‑dimethylformamide. The concentration of the free amine impurity, generated by adventitious Boc hydrolysis, is monitored by ion-pair HPLC and held below 0.2% area prior to shipment.
The synthesis route assembling the cis-amino alcohol scaffold relies on a late-stage Boc installation conducted at −10 to −5 °C in a 50 L De Dietrich glass-lined reactor fitted with a retreat-curve impeller and an external half-pipe jacket circulating brine at −20 °C. Maintaining this cryogenic window is mandatory because the N‑Boc derivative undergoes detectable ring-chain tautomerisation if the reaction mass warms above 0 °C before complete quench, generating up to 3% of a pyrrolidine ring-opened by‑product that co‑crystallises and degrades chiral purity. The quenching protocol uses pre‑chilled 2 M aqueous ammonium chloride delivered via a dosing pump at 0.5 L·min⁻¹ to avoid a thermal excursion beyond +2 °C.
Process safety evaluations performed on a Mettler‑Toledo RC1e reaction calorimeter reveal that the acid‑promoted removal of the Boc group from (3S,4S)-3-amino-4-hydroxypyrrolidine-1‑carboxylic acid tert‑butyl ester is accompanied by an exotherm of 45 kJ·mol⁻¹ of substrate when employing 20% v/v trifluoroacetic acid in dichloromethane. The liberated amino alcohol contains a β‑amino‑α‑hydroxy motif that is susceptible to acid‑catalysed dehydration, leading to an enamine intermediate that reprotonates with partial epimerisation at C‑4. Chiral HPLC monitoring of stressed samples indicates a epimerisation half‑life t1/2 of approximately 8 h at 20 °C that collapses to 45 min at 30 °C. Consequently, the deprotection step is run with an internal temperature setpoint of 0–5 °C and a jacket temperature of −15 °C, requiring a heat transfer coefficient U of at least 150 W·m⁻²·K⁻¹ to maintain isothermal conditions during the acid addition phase.
In vessels larger than 50 L, the specific jacket surface‑area‑to‑volume ratio drops below 2.0 m⁻¹, and the Reynolds number for the retreat‑curve impeller at 120 rpm falls into transitional flow, reducing the vessel‑side heat transfer film coefficient. A production campaign that attempted a 100 L scale‑up experienced a 7 °C temperature overshoot during TFA charge, resulting in 8.2% of the (3R,4S) diastereomer detected in the isolated crude. The diastereomer could not be reduced below 1.5% by recrystallisation and required flash chromatography on silica gel with a gradient of methanol in dichloromethane, adding 18 h to the work‑up and lowering the overall yield from 82% to 63%. This thermal sensitivity imposes an effective scale ceiling under batch processing as long as the deprotection is carried out in a homogenous TFA‑DCM medium; alternative heterogeneous cleavages with polymer‑bound sulfonic acids are under evaluation but introduce mass‑transfer limitations that reduce the effective reaction rate.
When the free base of (3S,4S)-3-amino-4-hydroxypyrrolidine is isolated after Boc cleavage and neutralisation, its storage stability degrades rapidly at relative humidity above 60%. The primary amine undergoes carbamate formation with atmospheric CO2 and the hydroxyl group facilitates water absorption that triggers a retro‑aldol‑type decomposition, producing a volatile aldehyde detectable by GC‑MS at m/z 72. For this reason, the N‑Boc protected form is supplied as the recommended stable intermediate, and downstream coupling steps are designed to retain the Boc group until after peptide bond formation.
The (3S,4S) configuration specifically places the hydroxyl hydrogen in proximity to the backbone carbonyl of a P2 proline surrogate, enabling a hydrogen‑bond network with the Ser‑195 side chain of the target protease as evidenced by published co‑crystal structures of related pyrrolidine‑based inhibitors. Replacement with the (3R,4R) enantiomer, an off‑white solid with [α]D20 +22° (c 1.0, MeOH) and melting range 96–103 °C, abolishes this interaction and results in a 35‑fold loss in factor Xa inhibitory potency (measured at 200 µM substrate concentration) relative to the (3S,4S) scaffold. Consequently, batches of the (3R,4R) material are typically relegated to negative control synthesis or used in metabolic stability comparisons where chirality‑dependent cytochrome P450 binding profiles are mapped. The (3S,4R)‑trans isomer, bearing an anti relationship between the amine and hydroxyl, displays an even shorter Boc‑removal half‑life of 6.5 h under the standard 20% TFA/DCM condition at 25 °C, attributed to anchimeric assistance from the syn‑axial hydroxyl when the ring adopts a C‑exo envelope conformation. This accelerated deprotection often leads to premature amine exposure during solid‑phase peptide couplings on 2‑chlorotrityl chloride resin, causing premature cleavage and sequence deletion.
| Compound | [α]D20 (c 1, MeOH) | t1/2 Boc removal† | Melting point (°C) | Orthogonal handle |
|---|---|---|---|---|
| (3S,4S)-Boc | −22° | 8.0 h | 95–102 | Boc removed under acidic conditions |
| (3R,4R)-Boc | +22° | 8.2 h | 96–103 | Boc removed under identical acid regime |
| (3S,4S)-Cbz | −18° | N/A (Pd/C, H2) | 78–84 | Cbz stable to TFA; deprotected by hydrogenolysis |
| (3S,4R)-Boc | +5° | 6.5 h | 112–118 (dec.) | Boc removed with accelerated kinetics |
† Deprotection half-life measured in 20% v/v TFA/DCM at 25 °C, monitored by chiral HPLC disappearance of starting material.
In fragment‑based assembly of renin inhibitors, the (3S,4S)-Boc intermediate is linked through its pyrrolidine nitrogen after Boc deprotection to a P3–P1 aldehyde warhead via reductive amination with NaBH(OAc)3 in dichloromethane containing 3 Å molecular sieves. Yields for this transformation, conducted on a 5 mmol scale in anhydrous DMF, reach 87% after chromatography on silica gel using 5% methanol in dichloromethane. Residual moisture in the solvent must be held below 50 ppm by Karl Fischer titration to avoid premature Boc cleavage during the reductive amination. The N‑Boc‑protected precursor also undergoes clean Mitsunobu inversion at C‑4 if an alternative stereochemistry is required, with diisopropyl azodicarboxylate and triphenylphosphine in tetrahydrofuran at 0 °C yielding the (3S,4R) acetate with >95% diastereoselectivity.
Thermogravimetric analysis of (3S,4S)-3-amino-4-hydroxypyrrolidine‑1‑carboxylic acid tert‑butyl ester reveals a mass loss of 0.3% upon heating to 120 °C, consistent with the specified moisture content. However, exposure of the solid to 75% relative humidity at 25 °C in an accelerated stability chamber results in a measurable increase of free amine content from 0.1% to 1.8% over 14 days, attributed to surface‑catalysed hydrolysis of the carbamate. The compound is therefore packaged in amber Type III glass bottles under a positive pressure of argon, sealed with PTFE‑lined polypropylene caps, and secondary‑bagged in aluminium‑laminated foil pouches containing a silica gel desiccant sachet. For long‑term storage, the recommended condition is 2–8 °C with protection from light and moisture. Redrying under vacuum (<10 mbar) at 30 °C for 4 h restores water specification without detectable racemisation, as shown by chiral HPLC before and after drying. Shipment validation data confirm that product integrity is maintained for 72 h at ambient temperatures not exceeding 30 °C, allowing global cold‑chain‑free distribution of research‑grade quantities.
