| HS Code | 433984 |
| Chemical Name | N-acetyl-L-proline |
| Iupac Name | (2S)-1-acetylpyrrolidine-2-carboxylic acid |
| Cas Number | 68-95-1 |
| Molecular Formula | C7H11NO3 |
| Molecular Weight | 157.17 g/mol |
| Smiles | CC(=O)N1CCCC1C(=O)O |
| Inchi | InChI=1S/C7H11NO3/c1-5(9)8-4-2-3-6(8)7(10)11/h6H,2-4H2,1H3,(H,10,11)/t6-/m0/s1 |
| Appearance | white crystalline powder |
| Melting Point | 113-115 °C |
| Optical Rotation | [α]20/D = -109° (c = 1 in water) |
| Solubility | soluble in water, ethanol, and dimethyl sulfoxide |
| Storage Conditions | store in a cool, dry, well-ventilated area, keep container tightly closed |
As an accredited N-acetyl-L-proline factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | White crystalline powder, 25 g per bottle, sealed in amber glass with desiccant, labeled with purity and handling precautions. |
| Container Loading (20′ FCL) | Load N-acetyl-L-proline in sealed drums on pallets, securely lashed in a 20′ FCL, protected from moisture and heat. |
| Shipping | N-acetyl-L-proline should be shipped in sealed, moisture-proof containers at ambient temperature, protected from heat, light, and humidity. It is generally non-hazardous, but include relevant documentation and SDS. Ensure proper labeling and customs declaration for international transport, avoiding contact with incompatible materials. |
| Storage | Store N-acetyl-L-proline in a tightly sealed container in a cool, dry place, protected from light and moisture. Refrigeration or freezing is recommended for long-term storage to maintain stability. Keep away from strong oxidizing agents and incompatible materials. Allow the container to equilibrate to room temperature before opening to prevent condensation. |
| Shelf Life | Store tightly sealed in a cool, dry, dark place. Stable for up to three years under these conditions. |
In multi-step manufacturing campaigns for proline-containing peptidomimetic active pharmaceutical ingredients, N-acetyl-L-proline is introduced as a protected chiral pool fragment that retains the L-configuration at the proline C-2 position while preventing premature acylation at the secondary amine. The compound is not handled as a finished-drug excipient; its control requirements follow ICH Q7 for active pharmaceutical ingredient starting materials and intermediates, with residual solvent testing aligned to ICH Q3C and USP <467>; typical limits for a process solvent such as ethyl acetate remain at 5000 ppm and for methanol at 3000 ppm unless a product-specific monograph imposes a tighter value. In a deprotection sequence intended to liberate L-proline for downstream coupling, the material is charged into 6 M HCl at 10–20 volumes per kilogram of N-acetyl-L-proline and held at reflux for 6–12 h; the resulting hydrolysate contains L-proline hydrochloride and acetic acid. Neutralization with 10 M NaOH to pH 6.5–7.0 is followed by vacuum concentration and crystallization from ethanol or acetone to isolate L-proline, while sodium acetate and sodium chloride remain partially in the mother liquor. When the acetyl group is retained, N-acetyl-L-proline is pre-activated with 1.05–1.15 molar equivalents of carbonyldiimidazole or an equivalent coupling reagent relative to the free amine of the growing intermediate, then coupled in a water-miscible solvent at 0–5°C to suppress racemization. The downstream products supplied from this route include proline-containing angiotensin-converting enzyme inhibitor intermediates and alanyl-proline dipeptide fragments used in peptidomimetic APIs. Residual water in the coupling step must remain below 0.1% w/w by Karl Fischer titration; higher water levels convert activated carbonyl intermediates into hydrolytically degraded carboxylate species and reduce coupling yield.
When N-acetyl-L-proline is used as a chiral resolving acid for racemic primary amine intermediates, the process window is defined by the crystallization trajectory after supersaturation release, not by the initial salt formation. Manufacturing of the resolved amine must follow ICH Q7 section 7.3 for critical process parameter documentation, with optical rotation determined by USP <781> and chiral purity by USP <621> or Ph.Eur. 2.2.29. The resolving acid is charged at 0.5–0.8 molar equivalents per mole of total racemic amine; full neutralization at 1.0 molar equivalent typically co-crystallizes the more soluble diastereomeric salt and reduces the enantiomeric excess of the isolated salt. In a representative production-scale crystallization, the racemic amine is dissolved in isopropanol/water 85:15 v/v at 40–50°C, and N-acetyl-L-proline is added as a solid or aqueous solution. After clarified filtration, the batch is cooled at 0.2–0.5°C/min to 15–20°C; seed crystals of the desired diastereomeric salt are introduced at 35–38°C at 0.1–0.5 wt% of theoretical yield to avoid spontaneous nucleation. Filtration and reslurry in the same solvent system are used to raise optical purity; published optical purity data for specific amine substrates is limited, and each substrate requires a polymorph and solubility screening program. Cooling rates above 0.5°C/min produce oiling-out and mixed diastereomer occlusion, which are the primary failure modes observed on production-scale crystallizers. The isolated salt is decomposed with a mild inorganic base to release the free amine, followed by extraction and distillation. Terminal product types include enantiopure primary amine intermediates used in chiral API manufacture, particularly for central nervous system and cardiovascular drug leads. The resolving agent must be kept away from strong mineral acids because N-acetyl-L-proline undergoes acid-catalyzed amide hydrolysis, generating L-proline and acetate and changing the salt stoichiometry.
| Process control point | Standard/method | Operational range | Observed failure mode |
|---|---|---|---|
| Cooling ramp after seed | ICH Q7 section 7.3 | 0.2–0.5°C/min | Oiling-out, mixed diastereomer occlusion |
| Seed addition temperature | ICH Q7 section 7.3 | 35–38°C | Uncontrolled nucleation and polymorph contamination |
| Seed loading | ICH Q7 section 7.3 | 0.1–0.5 wt% of theoretical yield | Oiling-out at lower seed mass |
| Specific optical rotation | USP <781> | Product-specific | Wrong enantiomer enrichment |
| Chiral purity | USP <621> | Product-specific | Enantiomeric impurity above limit |
Solid-phase peptide synthesis campaigns requiring an N-terminal N-acetylproline residue couple N-acetyl-L-proline directly onto the deprotected N-terminal amine of a resin-bound peptide; no subsequent Fmoc deprotection cycle is run for that residue because the acetyl group remains as a permanent cap. Specification setting for the resulting peptide API follows ICH Q6A, with chromatographic purity measured according to USP <621> or Ph.Eur. 2.2.29. The addition ratio is set at 2.0–4.0 molar equivalents of N-acetyl-L-proline relative to the free amine loading on the resin, with activation by 1.0–1.2 molar equivalents of HOBt and 1.0–1.2 molar equivalents of DIC in anhydrous DMF or NMP. Low-loading Wang or Rink amide resins at 0.3–0.6 mmol/g are preferred because higher loadings increase interchain aggregation and slow coupling of the N-acetylproline residue; resin swelling in DMF is controlled at 8–12 mL/g dry resin. Pre-activation is held for 3–5 min at 0–5°C before transfer to the solid support, and coupling proceeds for 1–2 h at 20–25°C. Incomplete coupling is capped with acetic anhydride/pyridine to avoid deletion sequences. Between base-mediated Fmoc removal and coupling, the resin is washed with 6–8 column volumes of DMF to remove residual piperidine; residual piperidine deprotonates the activated carboxylate and accelerates racemization at the proline α-carbon. Terminal product types include N-acetylproline-capped peptide APIs, antimicrobial peptides, and peptide substrates for diagnostic enzyme assays. The main operational boundary is residual water in DMF; values above 0.05% w/w by Karl Fischer reduce activation efficiency and produce truncated peptide impurities.
In leave-on cosmetic formulations such as moisturizing serums and barrier creams, N-acetyl-L-proline is added as a low-molecular-weight skin conditioning humectant; the INCI name N-Acetyl-L-Proline is listed in the European Commission CosIng database without an Annex II entry under EC 1223/2009. Manufacturing of the finished formula must follow ISO 22716:2007 good manufacturing practice, and the responsible person must maintain a product safety assessment under Article 10 of EC 1223/2009; for U.S. distribution, facility registration under 21 CFR Part 710 and product listing under 21 CFR Part 720 apply. The formulation development window is 0.05–0.5 wt% of the total formulation in leave-on products, with rinse-off formulations falling below 1.0 wt%; product-specific safety assessment may justify adjustments. The ingredient is dissolved in the aqueous phase at 35–45°C before addition of thickening polymers; the final pH is adjusted to 5.0–6.0 with 10% w/w citric acid or 10% w/w sodium hydroxide. Microbial quality is tested according to USP <61> and USP <62>; when water activity exceeds 0.6, preservative efficacy testing under ISO 11930:2019 is applied. Compatibility testing under ISO 22716:2007 change control is required when strong oxidizing agents or highly cationic polymer systems are present, because these systems can induce phase separation or pH drift in aqueous humectant blends. Terminal product types include oil-in-water moisturizing creams, hyaluronic acid combination serums, barrier-repair lotions, and after-sun hydrogels. Efficacy data on skin hydration is formulation-specific and must be generated under applicable claims-support requirements; published data for this specific configuration is limited.
For chiral ligand manufacture, N-acetyl-L-proline is converted to N-acetyl-L-prolinol through carboxylic acid reduction; subsequent deacetylation liberates L-prolinol, which is a precursor for oxazaborolidine catalysts and prolinol-derived ligands. The operation is governed by REACH registration for the starting material and reduced intermediate, with manufacturing traceability maintained under ISO 9001:2015; if the chiral ligand is used in an active pharmaceutical ingredient route, starting material controls under ICH Q11 apply. In the reduction step, the N-acetyl-L-proline is charged into anhydrous THF at 0–5°C, and 1.5–2.0 molar equivalents of borane-tetrahydrofuran complex are added at a rate that maintains the internal temperature below 5°C. Peroxide-free THF is mandatory; BHT-stabilized solvent is acceptable only after peroxide analysis by iodometric titration or ASTM E298. The borane complex reacts with the free carboxylic acid to form the corresponding primary alcohol; residual water in the solvent or substrate above 0.1% w/w by Karl Fischer titration consumes borane and accelerates exothermic hydrogen evolution. After complete addition, the mixture is aged for 2–4 h at 20–25°C, then quenched with methanol at ≤10°C; the quenched mixture is acidified to dissolve boron salts and extracted with a chlorinated or ethereal solvent. The isolated N-acetyl-L-prolinol is deacetylated under acidic or alkaline hydrolysis to yield L-prolinol before condensation with arylboronic acids to form oxazaborolidine catalysts. Terminal product types include L-prolinol-derived chiral ligands, CBS oxazaborolidine catalysts used in asymmetric ketone reduction, and N-acetyl-L-prolinol itself as a protected amino alcohol intermediate. Residual boron in the recovered prolinol is removed by aqueous workup and distillation; material exceeding the downstream catalyst purity specification is rejected because boron-containing contaminants interfere with subsequent metal complexation and reduce batch-to-batch catalyst reproducibility.
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N-Acetyl-L-proline, CAS 68-95-1, C7H11NO3, molecular weight 157.17 g/mol, is the N-terminal acetylated derivative of L-proline in which the secondary amine is converted to an acid-stable tertiary amide. The substance is supplied as a white to off-white crystalline powder with a reported melting range of 116–118 °C and a specific rotation in water typically specified between -86.0° and -89.0° at 589 nm. No universal pharmacopoeial or ISO model designation exists for this chemical entity; supplier model codes are proprietary, and procurement should therefore reference CAS 68-95-1, HPLC assay, enantiomeric purity, free L-proline, residual solvents, water, and residue on ignition. The acetyl group is stable to piperidine and trifluoroacetic acid under conventional solid-phase peptide synthesis conditions, which positions the compound as a capping or terminal building block rather than a temporary amine protector. The C-terminal carboxyl remains available for activation, while the pyrrolidine ring remains intact for downstream chiral intermediate chemistry.
Representative release limits for technical-grade N-acetyl-L-proline are directed at the contaminants most likely to interfere with downstream coupling chemistry. The following matrix summarizes the controls commonly applied on commercial certificates of analysis.
| Parameter | Control limit | Test method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual |
| Assay by HPLC, anhydrous basis | ≥ 98.0% area | C18 RP-HPLC, detection 210 nm |
| Enantiomeric purity | ≥ 99.0% L-isomer | Chiral HPLC with validated D/L separation |
| Free L-proline | ≤ 0.3% | Chiral HPLC or derivatization HPLC |
| Specific rotation | -86.0° to -89.0° (c=1, water) | Polarimetry at 589 nm |
| Melting range | 116–118 °C | Ph. Eur. 2.2.14 |
| Water | ≤ 0.5% | USP ⟨921⟩ Karl Fischer Method Ia |
| Residue on ignition | ≤ 0.1% | USP ⟨281⟩ |
| Heavy metals | ≤ 10 ppm | USP ⟨231⟩ or ICH Q3D elemental impurity assessment |
| Residual acetic acid | ≤ 0.5% | Headspace GC per Ph. Eur. 2.4.24 |
The free L-proline limit is not a chromatographic convenience. Residual secondary amine competes with the intended coupling substrate in active-ester or carbodiimide-mediated condensations; in automated synthesis, free proline contamination above 0.3% can generate truncated sequences or cap products that coelute with the target peptide at 210 nm on C18 columns. The optical rotation window is a release gate for enantiomeric integrity because values outside -86.0° to -89.0° indicate D-enantiomer contamination or incomplete acetylation. For water-sensitive coupling routes, material with Karl Fischer water above 0.5% is pre-dried at 40 °C under 1 kPa for 4 h before use.
In headspace GC release testing, residual solvents are assessed against ICH Q3C class limits. Ethyl acetate and methyl tert-butyl ether are class 3 solvents with recommended residual limits of 5000 ppm each. Methanol, if present as a crystallizing solvent, falls under ICH Q3C class 2 with a limit of 3000 ppm. Residual ethyl acetate is controlled because it can react under strongly basic coupling conditions, and residual methanol can compete as a nucleophile during amino acid activation.
In a standard Schotten-Baumann acetylation, L-proline is dissolved in aqueous sodium hydroxide and cooled to 0–5 °C before acetic anhydride is charged over 2–3 h. The pH is maintained between 9.0 and 10.5; excursions above 10.5 are associated with increased racemization, and published data for this specific configuration is limited, requiring chiral HPLC confirmation for each batch. After acetylation, the mixture is acidified to pH 2.0–2.5, extracted with ethyl acetate or methyl tert-butyl ether, washed with saturated sodium chloride, dried, and concentrated below 45 °C. Isolation by crystallization from ethyl acetate/hexane followed by vacuum drying yields the crystalline powder. Residual water and acetic acid are the principal release contaminants because both interfere with subsequent coupling chemistry; acetic acid consumed during activation can reduce coupling efficiency by neutralizing basic activators such as DIPEA or N-methylmorpholine.
Substitution of N-acetyl-L-proline for Fmoc-L-proline in solid-phase peptide synthesis is a chain-terminating event because the acetyl amide is stable to 20% piperidine in DMF and to TFA cleavage mixtures containing 95% TFA, 2.5% water, and 2.5% triisopropylsilane. Fmoc-L-proline, CAS 71989-31-6, is removed by piperidine within 10–20 min at room temperature and is therefore used for temporary protection. N-Acetyl-L-proline is used deliberately when a permanent N-terminal proline cap is required, as in peptide hormone analogs or in structure-activity studies where a free secondary amine at proline would alter receptor binding or degradation. In solution-phase synthesis, the free carboxyl is activated as the acid chloride, N-hydroxysuccinimide ester, or mixed anhydride; activation with isobutyl chloroformate and N-methylmorpholine at -15 °C typically gives low epimerization, but the product should be used immediately to prevent oxazolone formation. The acetyl group is not removed by hydrogenolysis or by TFA; its removal requires forcing acid hydrolysis such as 6 N HCl at 110 °C for 24 h, which also hydrolyzes the peptide backbone. This irreversible protection profile is the central difference from N-Boc-L-proline, CAS 15761-39-4, and N-Cbz-L-proline, CAS 1148-11-4.
Because the selection of a protected proline derivative is governed by downstream deprotection conditions, the following comparison is used during route design.
| Parameter | N-Acetyl-L-proline | L-Proline | N-Boc-L-proline | N-Cbz-L-proline |
|---|---|---|---|---|
| CAS | 68-95-1 | 147-85-3 | 15761-39-4 | 1148-11-4 |
| Molecular weight | 157.17 g/mol | 115.13 g/mol | 215.25 g/mol | 249.26 g/mol |
| N-terminal protection | Acetyl, acid-stable | Unprotected secondary amine | tert-Butoxycarbonyl, acid-labile | Benzyloxycarbonyl, hydrogenolysis-labile |
| Removal conditions | Forcing hydrolysis with 6 N HCl at 110 °C | Not applicable | TFA or HCl/dioxane at 0–25 °C | H2 over Pd/C at 1–3 bar |
| Typical use | Permanent N-terminal cap, chiral intermediate | Direct peptide incorporation, organocatalysis | Temporary protection in Boc/Bzl peptide synthesis | Temporary protection with orthogonal removal |
| Key limitation | Acetyl cannot be removed under standard SPPS deprotection | Secondary amine can react during activation | Acid-labile; deprotection generates TFA salt | Hydrogenolysis may be incompatible with sulfur-containing substrates |
N-Boc-L-proline is removed by TFA or HCl/dioxane at 0–25 °C, whereas N-Cbz-L-proline is removed by hydrogenolysis over Pd/C at 1–3 bar hydrogen. N-Acetyl-L-proline survives both TFA and hydrogenolysis; this stability is advantageous in downstream acidic workups but excludes the product from routes requiring temporary secondary-amine release. L-Proline itself remains the least sterically hindered and most hydrophilic building block, but its unprotected secondary amine can undergo side reactions during activation. In organocatalysis, L-proline participates in enamine-mediated aldol reactions; N-acetyl-L-proline does not form the required enamine and is not a substitute. Published data for direct comparative performance in every coupling system is limited, and route selection should be confirmed by process HPLC monitoring for epimerization.
Thermal analysis of N-acetyl-L-proline typically shows the reported melting endotherm near 116–118 °C; decomposition at higher temperatures is supplier-dependent, and published data for this specific configuration is limited. The material should be dried below 60 °C to avoid discoloration and partial amide hydrolysis in lots containing residual acetic acid. Vacuum drying at 40 °C and ≤1 kPa for 4–8 h reduces Karl Fischer water below 0.2% when open handling has exceeded 60% relative humidity. The compound is stored in closed, light-resistant containers at 15–25 °C; retest intervals of 24 months are common on certificates of analysis, but formal ICH stability data are not available for all packaging configurations. Aqueous solutions at neutral pH are stable for short-term use, but prolonged exposure to pH above 12 or to strong mineral acid at elevated temperature hydrolyzes the acetyl group to release L-proline. The product is incompatible with strong oxidizing agents and should not be combined with amine-based additives in storage because residual acid or activated ester formation can generate process-related impurities.
The compound is readily soluble in water and lower alcohols, moderately soluble in ethyl acetate, and nearly insoluble in n-heptane. In peptide synthesizers, 0.4 M solutions in DMF are typically prepared for automated coupling; the solution should be kept above 15 °C to avoid line precipitation. For activation with HATU, 2.0 equiv of DIPEA is used, with preactivation for 2–3 min at 0–5 °C before amine addition. This protocol reduces oxazolone formation when coupling to hindered amines; chiral purity is confirmed by sampling the reaction mixture after 30 min and analyzing by chiral HPLC with baseline resolution of D- and L-proline derivatives. In analytical development, N-acetyl-L-proline is also used as a retention-time marker and reference standard for monitoring acetylated proline impurities in peptide APIs. Calibration solutions are prepared in water/acetonitrile 90/10 v/v at 0.1–1.0 mg/mL and injected on C18 columns with UV detection at 210 nm.
For the process chemist, the central distinction between N-acetyl-L-proline and other protected proline building blocks is the permanence of the acetyl group. It permits acidic workup, ion-exchange chromatography, and TFA-based side-chain deprotection without loss of protection at the proline nitrogen, but it prevents subsequent chain elongation at that nitrogen. It is specified when the target molecule contains an N-terminal acetylated proline residue or when a proline derivative is needed as a chiral acid intermediate with the secondary amine blocked. Reaction monitoring is performed by reversed-phase HPLC at 210 nm with mass confirmation because the acetylated and unacetylated proline species can show only minor retention differences on C18 columns. Without a free secondary amine, N-acetyl-L-proline is unsuitable for enamine-mediated organocatalysis, but its acid-stable amide and free carboxyl make it a defined, monitorable intermediate in peptide and pharmaceutical synthesis.
