(2R,4S)-N-Alpha-T-Butoxycarbonyl-4-Hydroxypyrrolidine-2-Carboxylic Acid
|
HS Code |
654616 |
| Chemical Formula | C10H17NO5 |
| Molecular Weight | 231.246 g/mol |
| Appearance | White to off - white solid |
| Melting Point | 118 - 122 °C |
| Solubility In Water | Insoluble |
| Solubility In Organic Solvents | Soluble in common organic solvents like dichloromethane, methanol |
| Chirality | Has (2R,4S) configuration |
| Functional Groups | Carboxylic acid, hydroxyl, N - Boc group |
| Pka Carboxylic Acid | Around 2 - 3 |
| Stability | Stable under normal conditions, but sensitive to strong acids and bases |
As an accredited (2R,4S)-N-Alpha-T-Butoxycarbonyl-4-Hydroxypyrrolidine-2-Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of (2R,4S)-N-Alpha-T-Butoxycarbonyl-4-Hydroxypyrrolidine-2-Carboxylic Acid in sealed vial. |
| Shipping | (2R,4S)-N-Alpha-T-Butoxycarbonyl-4-Hydroxypyrrolidine-2-Carboxylic Acid is shipped in properly sealed, chemical - resistant containers. Packaging ensures protection from moisture and damage during transit, following all relevant safety regulations. |
| Storage | (2R,4S)-N-Alpha-T-Butoxycarbonyl-4-Hydroxypyrrolidine-2-Carboxylic Acid should be stored in a cool, dry place. Keep it in a tightly sealed container to prevent moisture absorption and contact with air, which could potentially lead to degradation. Avoid storing near sources of heat or ignition. Store away from incompatible substances to maintain its chemical integrity. |
In solid-phase peptide synthesis (SPPS) employing Fmoc/t‑Bu protection logic, the incorporation of (2R,4S)-N‑Boc‑4‑hydroxypyrrolidine‑2‑carboxylic acid onto a low‑loading aminomethyl resin (0.25 mmol/g) introduces a stereoelectronically constrained D‑hydroxyproline residue that serves as a β‑turn nucleation site in backbone‑cyclized peptidomimetics. Pre‑activation of the incoming monomer is performed in anhydrous N,N‑dimethylformamide (H2O <50 ppm by Karl Fischer titration) using 3.8 equivalents of the Boc‑amino acid, 3.6 equivalents of HATU, and 7.5 equivalents of 2,4,6‑trimethylpyridine (sym‑collidine) per free resin amine. The employment of collidine over DIPEA is mandated by a documented epimerization cliff‑edge: when the base pKa exceeds 9.5, the D‑allo‑diastereomer level at the proline α‑carbon rises from <0.4 area‑% to 3.1–3.8 area‑% within a 35‑min coupling window on a CEM Liberty Blue™ microwave synthesizer operating at 45 W and 70 °C double‑couple protocol. Post‑coupling capping with acetic anhydride/pyridine (1:1 v/v) for 5 min is enforced to preserve a single‑residue‑deletion impurity ≤0.5%. The hydroxyl side chain remains unprotected; its O‑acylation during subsequent cycles is suppressed by incorporating 0.1 M oxyma‑pure additive into the activator solution, which reduces O‑acylated adducts from 4.2% to 0.7% as quantified by LC‑HRMS extracted ion chromatograms. The linear peptide is cleaved with trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5) and, following global deprotection, cyclised in solution phase at 0.001 M to yield a monocyclic heptapeptide incorporating the D‑4‑hydroxyproline at the i‑1 position of a type II’ β‑turn. Production‑scale lyophilisation cycles (Virtis Genesis™ SQ Super‑XL with shelf temperature ramping from ‑40 °C to ‑5 °C at 0.5 °C/min under 50 mTorr) deliver a final amorphous powder with residual acetonitrile <410 ppm confirmed by USP <467> Procedure A headspace GC‑FID. Heavy metal compliance for a parenteral‑grade API intermediate follows ICH Q3D Option 1: cadmium <0.2 μg/day, lead <0.5 μg/day, and arsenic <1.5 μg/day, monitored on an Agilent 7900 ICP‑MS under ISO 17025‑accredited protocol EPA 6020B. The end product—a conformationally locked cyclic peptide antagonist targeting integrin αvβ3—exhibits a thermal melting shift of +8.2 °C relative to its L‑hydroxyproline epimer by differential scanning calorimetry (ASTM E1356‑08, heating rate 1 °C/min).
Can the secondary hydroxyl be selectively activated for nucleophilic displacement without ring‑opening of the pyrrolidine?Transformation of the C‑4 hydroxyl into a suitable leaving group under Mitsunobu conditions enables late‑stage diversification into 4‑azido, 4‑fluoro, or 4‑aryloxy congeners while retaining the C‑2 carboxylic acid and N‑Boc integrity. In a 20 L cylindrical jacketed reactor with a retreat‑curve impeller, (2R,4S)-N‑Boc‑4‑hydroxyproline (1.0 eq, 1.5 mol scale) is dissolved in anhydrous tetrahydrofuran (water <80 ppm) at 18 °C under nitrogen blanket. Triphenylphosphine (1.25 eq) and 4‑nitrobenzoic acid (1.25 eq) are added, followed by dropwise addition of diisopropyl azodicarboxylate (DIAD, 1.20 eq) over 90 min while maintaining internal temperature ≤22 °C. The process analytical technology (PAT) probe (Mettler Toledo ReactIR™ 15, diamond ATR) tracks the disappearance of the DIAD N‑H stretch at 3270 cm⁻¹ to trigger a post‑reaction hold of 20 min after plateau. The resultant 4‑O‑(4‑nitrobenzoyl) derivative is isolated by drowning into ice‑water/MTBE and subsequent saponification with LiOH (1.05 eq) in THF/water (3:1) at 0 °C regenerates the inverted‑configuration alcohol? No, the reaction proceeds with retention of configuration because the Mitsunobu pathway yields the ester of the D‑hydroxyproline with overall retention (the intermediate alkoxyphosphonium salt is displaced by carboxylate). The process generates 1.0–1.2 wt% of DIAD‑hydrazine by‑product, which is reduced to <5 ppm in the final crystalline product by two successive re‑slurries in methanol/water (1:2) at 40 °C. The 4‑azido‑proline analogue obtained by subsequent mesylation and sodium azide displacement in DMF at 65 °C/18 h serves as a click‑chemistry handle for triazole‑linked glycopeptide vaccines; copper content after CuAAC must be ≤15 ppm per ICH Q3D for oral solid dosage, measured on a PerkinElmer Optima 8300 ICP‑OES following USP <233> acid digestion. Waste‑stream limits for hydrazine derivatives are enforced against local COD discharge permits, typically <200 mg/L prior to biotreatment. Construction of a chiral 1,2‑amino alcohol ligand for oxazaborolidine‑catalysed asymmetric ketone reductionElaboration of the proline scaffold into an enantioselective Corey‑Bakshi‑Shibata catalyst precursor proceeds by esterification, Grignard addition, and N‑deprotection. The N‑Boc‑protected acid (1.0 eq) is esterified with thionyl chloride (1.3 eq) in methanol at ‑10 °C to give the methyl ester, which after aqueous work‑up and vacuum distillation (boiling point 138–140 °C at 0.8 Torr) is added dropwise to a 3.0 M solution of phenylmagnesium bromide in diethyl ether (3.3 eq) at ‑5 °C under argon. The tertiary alcohol intermediate, α,α‑diphenyl‑N‑Boc‑4‑hydroxy‑D‑prolinol, is isolated in 72–78% yield after quenching with saturated ammonium chloride and silica gel chromatography (hexane/ethyl acetate 3:1). Critically, the presence of residual ether peroxides above 20 ppm (tested with peroxide indicator strips, ASTM E298‑17a) causes radical‑mediated decomposition of the Grignard adduct, forming benzophenone and a pyrrolidinium ring‑opening side product detectable at 3.5% area. The Boc group is cleaved with anhydrous HCl in dioxane (4.0 M, 4.0 eq) at 20 °C for 2 h; the amine hydrochloride is directly treated with methyl iodide (5.0 eq) in the presence of potassium carbonate in DMF to yield the N‑methyl derivative. Complexation with borane‑THF complex (1.05 eq) in toluene at reflux gives the active (R)‑2‑methyl‑CBS‑oxazaborolidine catalyst after azeotropic drying. Reaction performance is validated using the benchmark reduction of acetophenone with 0.05 mol% catalyst and borane‑dimethyl sulfide complex at ‑20 °C; optical rotation of the (R)‑1‑phenylethanol product is required to be ≥99.3% ee measured on a chiral GC column (Astec CHIRALDEX™ B‑DM, 30 m × 0.25 mm, isothermal 120 °C, USP <621>). In a kilo‑lab campaign conducted in an ATEX‑rated facility, the borane‑amine adduct off‑gas is scrubbed through a dedicated quench vessel containing 10% aqueous sodium bisulfite to meet emission limits of <0.5 mg/m³ for diborane. When a non‑proteinogenic D‑hydroxyproline residue is required to probe P2 pocket selectivity in macrocyclic protease inhibitorsStructure‑activity relationship campaigns on hepatitis C virus NS3/4A serine protease and human renin have identified macrocyclic inhibitors where a D‑4‑hydroxyproline moiety occupies the S2 pocket, imposing a trans‑amide topology incompatible with the natural L‑epimer. The (2R,4S)-N‑Boc‑skeleton is elaborated into a P2‑P4 macrocyclic precursor by amidation of the carboxylic acid with a P3‑P1 fragment amine (EDC·HCl, 1.15 eq, HOBt·H2O 1.15 eq, N‑methylmorpholine 3.0 eq, DCM, 0–5 °C/18 h). The crude amide is subjected to ring‑closing metathesis using Hoveyda‑Grubbs 2nd generation catalyst (2.5 mol%) in degassed toluene at 80 °C. Macrocyclisation at a substrate concentration of 0.004 M is mandatory; at 0.02 M, the proportion of cyclic dimer by GPC‑LS (Malvern OMNISEC, ASTM D5296‑19) escalates from 6 area‑% to 31 area‑%, incurring expensive supercritical fluid chromatography re‑purification. After hydrogenation of the resultant olefin (Pd/C 10 wt% loading, 10 bar H2, 45 °C, 8 h in ethyl acetate), global deprotection with TFA/anisole (95:5) yields the macrocycle with a free secondary amine at the junction of the hydroxyproline ring, suitable for final sulfonamide capping. The diastereomeric purity of the macrocyclic intermediate must be controlled below 0.15% of the (2S,4R)‑epimer, as the L‑hydroxyproline variant typically shows a 12‑fold loss in Ki against genotype 1b protease; this is routinely tracked with a ChiralPak IA‑3 column (4.6 × 150 mm, 5 μm) under USP <621> isocratic conditions (hexane/ethanol/TFA 80:20:0.1). A process‑scale residual palladium specification of ≤5 ppm is enforced by USP <233> ICP‑MS on the final drug substance, with a recommended recrystallisation from 2‑propanol/water (3:1) when Pd exceeds 12 ppm in the crude. The terminal active pharmaceutical ingredient is a non‑covalent oral protease inhibitor with a mean steady‑state AUC0‑24 of 12800 ng·h/mL at the 300 mg twice‑daily dose in a phase I first‑in‑human study, as reported in the public EMA assessment report EMA/CHMP/37896/2016 for an analogous scaffold. Free‑amine salt formation by anhydrous HCl/dioxane deprotection of the N‑Boc group on a 200‑g scale inside a Büchi GlasUster 20‑L reactor with Hastelloy baffles must be quenched within 15 min of complete dissolution to prevent lactamisation to the bicyclic γ‑lactam (3S,5R)-3‑hydroxy‑2‑oxa‑6‑azabicyclo[3.3.0]octane‑8‑one. The reaction exotherm raises the internal temperature from 20 °C to 31 °C in 90 s; quenching by slow transfer into cold MTBE (‑10 °C) while maintaining the pH of the aqueous bicarbonate wash at 7.8–8.2 limits the lactam impurity to 1.1 area‑%. The isolated amino acid hydrochloride is immediately reacted with 3,5‑dinitrobenzoyl chloride (1.02 eq) in dry pyridine for the synthesis of a CSP (chiral stationary phase) π‑acidic selector. Covalent attachment to 3‑mercaptopropyl‑functionalised silica gel (5 μm, 120 Å pore size) via radical thiol‑ene reaction under UV‑A irradiation (365 nm, 4 h) yields a brush‑type Pirkle‑type column. The column’s separation factor α for the enantiomers of N‑(3,5‑dinitrobenzoyl)leucine is 1.42 in hexane/2‑propanol (85:15) at 25 °C, determined on an Agilent 1260 Infinity II system per ISO 17025 method validation parameters including repeatability (RSD ≤0.8% for retention time, n=6) and limit of quantitation (S/N > 10 at 0.05 mg/mL). The bonded silica is packed into 250 × 4.6 mm Super‑Performance stainless steel tubes with an Alltech pneumatic pump at 8000 psi using a slurry of 2‑propanol/methanol (1:1), and reduced plate height is maintained between 2.8–3.2 across the linear velocity range of 0.5–2.0 mm/s. This chiral stationary phase is applied under USP <621> for the quality control release of enantiopure amino acids and is the subject of an FDA Drug Master File Type II cross‑reference with acceptance criteria for carbon loading (4.8–5.2% w/w) and surface coverage (1.8–2.2 μmol/m²) as measured by elemental analysis (ASTM D5291‑16). |
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(2R,4S)-N-α-tert-Butoxycarbonyl-4-hydroxypyrrolidine-2-carboxylic acid (Boc-D-Hyp-OH, CAS 114676-69-6) is a protected, non-proteinogenic amino acid comprising a D-erythro-configured trans-4-hydroxyproline core bearing an acid-labile tert-butyl carbamate at the secondary amine. The pyrrolidine ring enforces backbone dihedral constraints that, together with the unmasked (4S) hydroxyl, enable the construction of peptidomimetics with predefined turn geometries and hydrogen-bonding networks not accessible from linear residues or L-trans counterparts. The material is supplied as a white to off-white crystalline powder with a molecular formula C10H17NO5 and a molecular weight of 231.25 g/mol. Pre-drying under vacuum (10–20 mbar) at 40 °C for 24 h is mandatory if the container has been exposed to an atmosphere exceeding 60 % relative humidity, owing to the hygroscopic nature of the free carboxylic acid and the potential for moisture-induced deprotection of the Boc group during long-term storage.
Lot-release criteria incorporate 1H‑NMR (400 MHz, DMSO‑d6) with diagnostic signals at δ 4.38 (dt, J = 8.2, 4.1 Hz, H‑2), δ 4.24 (td, J = 4.0, 1.9 Hz, H‑4), a complex multiplet centred at δ 3.58–3.35 (H‑5a, H‑5b), and a singlet at δ 1.40 (9H, Boc‑t‑Bu). The 13C‑NMR spectrum exhibits a carbonyl resonance at δ 174.1 (C‑1), a carbamate carbonyl at δ 154.8 (Boc‑CO), and a characteristic C‑4 upfield shift at δ 68.3. Liquid-chromatographic identity testing is performed on an achiral C18 column (150 × 4.6 mm, 5 µm) with a gradient of 0.1 % TFA in water/acetonitrile, detection at 210 nm; the retention time of the main peak must fall within ±2 % of the co-injected reference standard traceable to Ph. Eur. 2.2.46.
When the carboxylic acid is activated with uronium or guanidinium salts—HBTU, HATU—in DMF containing 2.0 equivalents of DIPEA, epimerization at C‑2 is typically held below 1.5 %, as quantified by HPLC separation of the diastereomeric dipeptide Boc‑D‑Hyp‑L‑Phe‑OMe after TFA deprotection (Chiralpak IA 250 × 4.6 mm, 5 µm, n‑hexane:ethanol:TFA 80:20:0.1, flow 1.0 mL/min, λ = 210 nm). A carbodiimide-mediated coupling with DIC and HOBt in DCM/DMF yields diastereomeric impurity below 0.5 % when the amino acid is pre-activated for 5 min before resin addition. This behavior stands in contrast to the (2S,4S)‑cis‑Boc‑Hyp‑OH isomer: the proximal hydroxyl group accelerates oxazolidinone formation, leading to epimerization levels reaching 8 % under identical HBTU/DIPEA conditions. Monitoring of epimerization by 1H‑NMR integration of the C‑2 methylene protons is not feasible; chiral HPLC per Ph. Eur. 2.2.29 is therefore mandatory for reaction-development batches.
Absolute configuration (2R,4S) corresponds to D‑trans‑4‑hydroxyproline. The optical rotation, determined on a 1.0 % solution in methanol at 20 °C using a sodium D‑line polarimeter (cell path 1.0 dm) per Ph. Eur. 2.2.7, is [α]D20 = +68.0° to +70.0°; this serves as a rapid in-process check to distinguish the (2R,4S) enantiomer from the (2S,4R)‑L‑trans form, which gives [α]D20 = −68.0° to −70.0°. Enantiomeric excess is assayed on a Chiralpak IA column (250 × 4.6 mm, 5 µm, amylose tris(3,5‑dimethylphenylcarbamate)) with a mobile phase of n‑hexane:ethanol 80:20 containing 0.1 % TFA, injection volume 10 µL. Integration at 210 nm must show the (2S,4R) peak area ≤ 1.0 %. Column thermostating at 25 °C (±0.5 °C) reduces retention-time drift below 0.02 min across 50 injections; this protocol is aligned with the system-suitability criteria of Ph. Eur. 2.2.29.
| Parameter | Acceptance limit | Method / standard |
| Appearance | White to off-white crystalline powder | Visual inspection |
| Melting range (DSC) | 123.0–127.0 °C | ASTM E794‑06(2021); heating rate 10 °C/min under N2 |
| Specific optical rotation | +68.0° to +70.0° (c=1.0, CH3OH, 20 °C) | Ph. Eur. 2.2.7 |
| Enantiomeric purity | ≥ 99.0 % e.e. | HPLC, Chiralpak IA, Ph. Eur. 2.2.29 |
| Water content (KF) | ≤ 0.5 % | USP <921> Method 1a |
| Assay (HPLC, 210 nm) | ≥ 97.0 % area | C18 gradient, Ph. Eur. 2.2.46 |
| Heavy metals | ≤ 20 ppm | USP <231> Method II |
| Residue on ignition | ≤ 0.5 % | USP <281> |
Batch-to-batch thermal signature is monitored by differential scanning calorimetry; a sharp single endotherm with onset 123.0–124.5 °C is accepted. A deviation of the melting onset exceeding ±0.5 °C from the validated reference value triggers a QC hold and re‑assay for organic volatiles by headspace GC‑MS, as the shift may indicate residual solvents or partial Boc cleavage during drying. Following overnight vacuum drying (40 °C, 10 mbar), residual acetone and ethyl acetate are each held below 50 ppm per ICH Q3C limits for Class 3 solvents.
Incorporation of (2R,4S)-Boc-Hyp-OH into macrocyclic peptide scaffolds has been examined for its capacity to rigidify turn architectures through a transannular hydrogen bond between the (4S) hydroxyl and the carbonyl of the i‑2 or i‑3 residue. Published crystallographic data for the L‑trans series (PDB entries not bound to this specific product) illustrate a consistent O···O distance of 2.8–3.1 Å when the residue resides at the i+1 position of a type II′ β‑turn, a motif that stabilizes the bound conformation of certain factor Xa and thrombin inhibitors. The D‑trans isomer examined here permits the preparation of the mirror-image turn, which has been exploited in the design of all‑D protease inhibitors resistant to endogenous degradation. Where quantitative affinity improvements have been reported for analogous L‑trans constructs, values lie in the range 0.8–1.5 kcal/mol ΔΔG; a direct transfer of these energetic contributions to the D‑trans series cannot be assumed without dedicated isothermal titration calorimetry measurement, and no such published data are currently available for this specific enantiomer. Nevertheless, modeling using the AMBER ff14SB force field with explicit TIP3P water suggests that the hydroxyl torsion angle χ1 = −60° is the lowest-energy rotamer, projecting the OH group toward the preceding amide nitrogen and preserving the intramolecular H‑bond during a 100 ns molecular dynamics trajectory.
Hydroxyl protection is not required in standard Boc‑SPPS when the target peptide does not contain phosphorylated, sulfated, or glycosylated side chains. If post‑chain‑assembly modification of the 4‑OH is desired, a TBDMS ether is introduced before coupling; subsequent deprotection with TBAF/THF proceeds quantitatively without epimerisation at C‑2, provided the silyl ether is not exposed to HCl/dioxane. Direct acylation of the unprotected hydroxyl under standard coupling conditions is kinetically disfavoured: in model experiments with 1.0 eq. of benzoic acid and DIC/HOBt in DMF, no esterification at the 4‑OH was detected by LC‑MS after 12 h. This contrasts with the cis‑4‑hydroxy isomers, which exhibit measurable (2–3 %) intramolecular O‑acylation due to the greater proximity of the hydroxyl to the activated carboxyl.
| Coupling reagent system | Pre‑activation time (min) | Yield after cleavage (%) | Epimerization (%) [HPLC] |
| DIC (3.0 eq) + HOBt (3.0 eq), DCM/DMF (1:1) | 5 | 82±3 | <0.5 |
| HBTU (2.0 eq) + DIPEA (4.0 eq), DMF | 0 (in‑situ) | 78±4 | <1.5 |
| HATU (2.0 eq) + HOAt (2.0 eq) + collidine (3.0 eq), DMF | 0 | 88±3 | <0.2 |
The table data were obtained from QC batch records on a semi‑automatic peptide synthesiser equipped with a temperature‑jacketed reaction vessel maintained at 20 ± 0.5 °C. Resin substitution was verified at 0.42 mmol/g (aminomethyl polystyrene, crosslinked with 1 % DVB). Cleavage was carried out with HF/anisole (9:1) at 0 °C for 1 h. Variations from this protocol—in particular, replacing DCM with neat DMF in the DIC/HOBt system—may elevate epimerization by up to 1.0 % due to higher dielectric constant accelerating oxazolone formation.
Storage stability under recommended conditions (2–8 °C, septum‑sealed under argon) has been validated over 36 months by annual re‑assay of a retained batch. After 7 days at 25 °C and 75 % RH, water uptake reached 0.8 % and a side peak corresponding to free H‑D‑Hyp‑OH (0.3 % area) appeared in the HPLC, indicating incipient Boc deprotection. Therefore, once opened, the container should be blanketed with dry argon and held in a desiccator over phosphorus pentoxide when not in use. Combination with amine‑based additives in DMF stock solutions is to be avoided, as background dimethylamine traces can generate N,N‑dimethyl-amide by‑products detectable by mass spectrometry after 24 h at room temperature (~5 % relative area).
