| HS Code | 458580 |
| Product Name | CBZ-DL-phenylalanine |
| Iupac Name | 2-[(benzyloxycarbonyl)amino]-3-phenylpropanoic acid |
| Cas Number | 3588-57-6 |
| Molecular Formula | C17H17NO4 |
| Molecular Weight | 299.32 g/mol |
| Synonyms | N-Carbobenzoxy-DL-phenylalanine; Z-DL-Phe-OH |
| Appearance | White to off-white crystalline powder |
| Melting Point | 103-105 °C |
| Purity | ≥98% (HPLC) |
| Storage Conditions | Store at 2-8°C, keep container tightly closed, protect from light |
| Solubility | Soluble in ethanol, methanol, DMF, DMSO; sparingly soluble in water |
As an accredited CBZ-DL-phenylalanine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 25 g of CBZ-DL-phenylalanine in a sealed amber glass bottle with tamper-evident cap and labeled safety information. |
| Container Loading (20′ FCL) | 20′ FCL loading: drum-packed CBZ-DL-phenylalanine, securely palletized and strapped, protected from moisture, with proper ventilation and labeling. |
| Shipping | CBZ-DL-phenylalanine ships as a non-hazardous chemical under standard conditions. Pack in sealed, moisture-resistant containers away from heat and incompatible substances. Avoid generating dust; use local exhaust ventilation. No special transport classification required, but ensure labeling and documentation match safety data sheet. |
| Storage | Store CBZ-DL-phenylalanine in a tightly sealed container, protected from light and moisture, in a cool, dry, well-ventilated area. Room temperature is generally suitable. Keep away from strong oxidizing agents and sources of ignition. Ensure the container is clearly labeled and inaccessible to unauthorized personnel to maintain purity and stability. |
| Shelf Life | Store tightly sealed, protected from light, at 2–8°C. Under these conditions, shelf life is typically 2–3 years. |
During multi-kilogram solution-phase synthesis of peptide drug intermediates, CBZ-DL-phenylalanine is introduced as a carbobenzoxy-protected acyl donor in isobutyl chloroformate-mediated mixed anhydride couplings. In 1,000–2,000 L glass-lined reactors, batch records show that moisture intrusion above 0.05% KF in dichloromethane reduces mixed anhydride formation and extends phase splits during the sodium bicarbonate wash. A representative charge ratio for Cbz-DL-Phe acid to amine hydrochloride is 1.00 : 1.00 mol, with N-methylmorpholine at 1.05–1.15 mol and isobutyl chloroformate at 0.95–1.05 mol, maintained between -15 °C and -5 °C for 15–30 min. The amino component is added as a pre-neutralized free amine or as the hydrochloride in the presence of a tertiary base; coupling is then warmed to 18–22 °C over 60–90 min and followed by 0.5 M HCl, 5% NaHCO3, and saturated NaCl washes. Vacuum distillation at ≤45 °C and crystallization from ethyl acetate/n-heptane yielded protected dipeptide esters such as Cbz-DL-Phe-Gly-OMe and Cbz-DL-Phe-Leu-OEt with HPLC area purity above 97.5% by USP <621>. REACH registration under EC 1907/2006, ICH Q7/Q11 supplier qualification, and residual solvent reporting under ICH Q3C apply. The terminal products are protected phenylalanine-containing dipeptide and tripeptide fragments for subsequent solution-phase elongation in peptide API manufacture; the racemic centre is processed only where downstream chiral separation or crystallisation-induced enrichment is part of the registered process. The operational boundary is strict exclusion of primary or secondary amines before activation, because premature aminolysis of the mixed anhydride consumes the reagent and shifts the mass balance below 80%.
Methyl ester formation from CBZ-DL-phenylalanine precedes enzymatic resolution because the free acid is a weak substrate for the alkaline protease. In a 2 L jacketed pH-stat vessel, Cbz-DL-Phe-OMe is charged at 0.20–0.40 mol/L in 0.1 M phosphate buffer at pH 7.5–8.0 and 30–40 °C; Alcalase 2.4 L FG loading is 2.0–5.0 g/L with 5–10 vol% DMSO to maintain substrate solubility. The reaction is titrated with 0.5 M NaOH to hold pH within ±0.1 unit; a pH overshoot beyond 8.5 accelerates non-selective background hydrolysis and reduces enantiomeric excess below 95%, which is a critical process failure. Termination at 45–50% conversion of the racemic ester, followed by phase separation of the remaining organic ester fraction from the hydrolyzed acid fraction, produces Cbz-L-phenylalanine in the aqueous phase after acidification to pH 2.0–2.5 with 6 M HCl. The terminal products are enantiopure Cbz-L-Phe-OH and Cbz-D-Phe-OMe; the latter is saponified to Cbz-D-Phe-OH under controlled 0.5 M NaOH at ≤25 °C. Compliance under ISO 9001:2015 supplier quality management and ICH Q7 when the resolved material enters a drug substance route is required; analytical identity is confirmed by chiral HPLC with Ph. Eur. 2.2.46 system suitability, and residual enzyme protein is specified at <0.1% (w/w) for pharmaceutical-grade material. Published data for the identical reaction using the free acid CBZ-DL-phenylalanine rather than the methyl ester are limited; process development work should therefore maintain ester activation to reach target ee above 98% after a single extraction.
For peptide intermediates containing thioether or thiol residues that poison palladium catalysts, Cbz removal by low-pressure hydrogenolysis is the preferred route only when the substrate is sulfur-lean. In standard hydrogenolysis of isolated CBZ-DL-phenylalanine-containing intermediates, 10% Pd/C (50–60% water wet) is charged at 5–10 wt% relative to substrate, with hydrogen pressure controlled at 0.2–0.4 MPa and jacket temperature at 25–35 °C in a 500 L Hastelloy C22 stirred autoclave. Hydrogen uptake of 1.0–1.5 equivalents per Cbz group, followed by plateau, is used as the reaction endpoint; over-reduction of aromatic rings is minimized by stopping hydrogen feed at ≤0.4 MPa and by avoiding prolonged agitation beyond 8 h. The post-reaction slurry is filtered through a 0.2 μm polypropylene depth filter pre-coated with 1–2 kg Celite per 10 kg substrate charge, then treated with activated carbon at 2–4 wt% for 2–4 h at 40–50 °C. Terminal products are the deprotected phenylalanine-containing peptide as acetate or free base after salt exchange; residual palladium is controlled according to ICH Q3D with an oral PDE of 100 μg/day, a parenteral PDE of 10 μg/day, and supplier intermediate specifications typically ≤10 ppm by USP <233>. Process safety compliance follows ATEX 2014/34/EU for hydrogen handling and ISO 9001:2015 for change control. The operational boundary for catalyst reuse is substrate-dependent: sulfur-containing thioether residues at >0.1 mol% deactivate the catalyst bed and require fresh catalyst charge per batch.
| Control point | Method/standard | Limit |
|---|---|---|
| Oral drug substance Pd PDE | ICH Q3D | 100 μg/day |
| Parenteral drug substance Pd PDE | ICH Q3D | 10 μg/day |
| Intermediate supplied to GMP route | USP <233> ICP-MS | ≤10 ppm |
| Pre-filtration load pH | pH meter | 4.5–8.0 |
For chromogenic chymotrypsin substrate manufacture, the N-benzyloxycarbonyl-protected p-nitrophenyl ester is produced by DCC/DMAP-mediated esterification in dry ethyl acetate/DMF. In 10:1 (v/v) ethyl acetate/DMF, Cbz-DL-Phe is charged at 1.00 mol, 4-nitrophenol at 1.00–1.10 mol, DCC at 1.10–1.30 mol, and 4-DMAP at 0.05–0.10 mol under nitrogen at 0–5 °C; the dicyclohexylurea precipitate is removed by filtration after 12–16 h at 2–8 °C. The organic phase is washed with cold 0.5 M HCl, 5% NaHCO3, and saturated NaCl; residual dicyclohexylurea and free 4-nitrophenol are reduced by recrystallization from toluene/n-heptane to yield Cbz-DL-Phe-ONp with HPLC purity ≥98.0% and water content <0.2%. The terminal product is used in chymotrypsin kinetic assays with detection at 400–410 nm, either as the racemic mixture for broad activity screening or as the enantiopure Cbz-L-Phe-ONp after resolution. For IVD reagent manufacturers, ISO 13485:2016 supplier controls and Ph. Eur. 2.2.46 or USP <621> chromatographic system suitability apply; residual solvents are reported under ICH Q3C, and residual dicyclohexylurea is limited to ≤0.1% (w/w) because it inhibits chymotrypsin at higher concentrations. The operational boundary is strict exclusion of water during DCC activation; moisture above 0.1% KF reduces ester yield below 85% and shifts impurity profiles toward N-acylurea.
Laboratory and pilot-plant records for Z-protected diamine-sensitive peptide intermediates show that acidolysis with 33 wt% HBr in glacial acetic acid is selected when hydrogenation cannot proceed because sulfur-containing residues poison the metal catalyst or when unsaturation elsewhere in the molecule contraindicates hydrogen addition. CBZ-DL-phenylalanine-containing peptides are dissolved in 33 wt% HBr/AcOH at 5–10 mL/g substrate, with reactor jacket held at 0–25 °C and reaction time limited to 2–6 h; the carbobenzoxy group fragments as benzyl bromide and carbon dioxide, while the peptide precipitates as the hydrobromide salt upon addition of cold diethyl ether at 8–12 times the reaction volume. The crude salt is filtered under nitrogen, washed with diethyl ether, and vacuum-dried at ≤30 °C to avoid ester exchange and hygroscopic degradation. Terminal products are phenylalanine-containing peptide hydrobromide salts that are converted to acetate or free base by ion exchange before subsequent coupling. Compliance under ICH Q3C for residual acetic acid and diethyl ether, USP <467> for residual solvent testing, REACH Annex II for downstream use communication, and ISO 9001:2015 for batch documentation applies. The critical operational boundary is acid-labile functionality: tert-butyl esters and glycosidic bonds are partially cleaved under these conditions; therefore the method is limited to peptide intermediates carrying acid-stable side-chain protections. If a peptide contains a readily oxidizable methionine residue, oxygen exclusion and the addition of dimethyl sulfide as scavenger at 0.5–1.0% (v/v) are required to prevent sulfoxide formation above 0.15%.
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CBZ-DL-phenylalanine, formally N-[(benzyloxy)carbonyl]-DL-phenylalanine (registry number 24222-45-4; molecular formula C17H17NO4; molar mass 299.32 g/mol), is supplied as a white to off-white crystalline powder with a typical initial HPLC purity of 98.0% by area normalization at 210 nm. Commercial research-grade models are cataloged as CBZ-DL-Phe-OH-25G, CBZ-DL-Phe-OH-100G, CBZ-DL-Phe-OH-500G, and CBZ-DL-Phe-OH-1KG; bulk material is packed in 25 kg fiber drums with double polyethylene liners under nitrogen. The compound dissolves in methanol, ethanol, ethyl acetate, and dimethylformamide; aqueous solubility remains below 1 mg/mL at 20 °C. The carbobenzoxy group is stable under mild basic conditions and common coupling conditions, but is removed by catalytic hydrogenolysis over palladium or by strong acids such as HBr in acetic acid. That protecting-group profile separates the Cbz derivative from the base-labile Fmoc analogue and the acid-labile Boc analogue.
A representative supplier certificate of analysis applies the following release criteria. No current EP, USP, or JP monograph for this exact racemic derivative has been published; the acceptance limits are therefore derived from general monograph methods and validated in-house HPLC procedures.
| Parameter | Acceptance criterion | Analytical method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual examination |
| Identification by infrared absorption | Spectrum concordant with reference | EP 2.2.24 |
| HPLC purity | ≥98.0% | C18 column, 210 nm, phosphate buffer pH 2.8: acetonitrile gradient |
| Individual related substance | ≤0.50% | Same HPLC method as above |
| Specific rotation [α]D20 | −0.5° to +0.5° | EP 2.2.7 |
| Loss on drying | ≤0.50% | 60 °C vacuum, 4 h |
| Residue on ignition | ≤0.20% | EP 2.4.16 |
| Heavy metals | ≤20 ppm | EP 2.4.8 |
Residual solvent testing by gas chromatography headspace according to EP 2.4.24 typically limits dichloromethane to ≤600 ppm, methanol to ≤3000 ppm, and ethyl acetate to ≤5000 ppm, consistent with class 2 solvent allowances when the material is used as a pharmaceutical intermediate. Elemental impurity screening by inductively coupled plasma mass spectrometry applies the ICH Q3D option 2A limits for oral drug products; palladium is controlled to ≤10 ppm when the material has been exposed to hydrogenation catalysis.
The racemic character of CBZ-DL-phenylalanine is expressed by an equimolar D- and L-enantiomer composition; a 1.0% solution in methanol at 20 °C exhibits specific rotation [α]D20 of 0.0° ± 0.5°. In peptide synthesis, the D-phenylalanine component generates diastereomeric intermediates when coupled to enantiopure amino acid esters. Coupling of CBZ-DL-phenylalanine with L-proline methyl ester through N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole in dichloromethane at 0–5 °C produces two diastereomers separated by 4.0–6.0 min on C18 reversed-phase HPLC at 220 nm. The D:L ratio in the product is controlled less by oxazolone racemization, because the starting acid is already racemic, than by the relative reaction rates of the two enantiomers; residual water influences the active-ester equilibrium and can increase N-acylurea side-product formation. Specifications for diastereomeric composition commonly require total related substances not more than 2.0%, with the target diastereomer area percentage set according to the downstream peptide synthesis route.
| Attribute | CBZ-DL-phenylalanine | CBZ-L-phenylalanine | Fmoc-DL-phenylalanine |
|---|---|---|---|
| Molecular formula | C17H17NO4 | C17H17NO4 | C24H21NO4 |
| Molar mass | 299.32 g/mol | 299.32 g/mol | 387.43 g/mol |
| Protecting group lability | Stable to mild base and trifluoroacetic acid; cleaved by hydrogenolysis or HBr/acetic acid | Same as DL derivative | Stable to acid; cleaved by secondary amines such as piperidine |
| Typical deprotection condition | H2 over 10% Pd/C at 1–4 bar or 33% HBr/acetic acid | Same as DL derivative | 20% piperidine in dimethylformamide, 20–25 °C |
| Stereochemical form | Racemic mixture | L-enantiomer | Racemic mixture |
| Main synthetic role | Diastereomer synthesis, chiral resolution studies, reference standards | Enantiopure peptide synthesis | Fmoc solid-phase peptide synthesis |
The Cbz group occupies a distinct position among N-protecting groups because its removal is orthogonal to both the acid-labile Boc group and the base-labile Fmoc group. The racemic Cbz derivative can therefore be carried through sequences that include trifluoroacetic acid treatment for Boc removal or piperidine treatment for Fmoc removal without loss of the carbobenzoxy group. This orthogonality is used in convergent peptide fragment condensation, where CBZ-DL-phenylalanine is coupled to a peptide fragment that contains Boc or Fmoc side-chain protection. The main limitation is release of the phenylalanine racemate as an unprotected N-terminal residue after hydrogenolysis, which cannot be used directly for enantiomerically defined peptide elongation unless the racemate is resolved by chiral crystallization or preparative chiral chromatography.
For chiral method transfer, CBZ-DL-phenylalanine is used as a system suitability standard to demonstrate baseline resolution on amylose tris(3,5-dimethylphenylcarbamate) or cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases. Under n-hexane/ethanol/formic acid 85:15:0.1 at 25 °C and a flow rate of 0.8 mL/min, the D- and L-enantiomers of the Cbz derivative elute with separation factors that depend on the column lot; published data for this specific configuration is limited, so resolution factors are established during method qualification. An area ratio outside 48.0:52.0 D:L indicates non-racemic material or enantiomeric enrichment introduced during synthesis or storage.
CBZ-DL-phenylalanine is also employed as a reference standard in impurity profiling of enantiopure CBZ-L-phenylalanine starting material. The D-enantiomer signal in chiral HPLC may be quantified against a 0.1% external standard; a racemic sample of CBZ-DL-phenylalanine provides a 50:50 D:L ratio for calibration of detector linearity across the range 0.05–2.0% area. In manufacturing release of CBZ-L-phenylalanine, the acceptance limit for the unwanted D-enantiomer is typically ≤0.5%; the racemic standard is therefore used to bracket the specification limit.
Production-scale coupling with CBZ-DL-phenylalanine has been documented in stirred glass-lined reactors with working volumes of 100–500 L. In one configuration, the acid is first converted to the N-hydroxysuccinimide ester in dichloromethane with dicyclohexylcarbodiimide at 0–5 °C. The dicyclohexylurea by-product, insoluble in dichloromethane, is removed by filtration through a 10 μm bag filter; incomplete filtration leads to downstream column backpressure and residual urea in the isolated peptide intermediate. The ester is transferred to a coupling vessel maintained at −5 to 0 °C to reduce diketopiperazine formation when proline or N-methyl amino acid residues are present. HPLC analysis of the coupling stream at 220 nm provides a reaction conversion of ≥95.0% before water quench. After phase separation, the organic layer is washed with 5% w/v sodium bicarbonate and 10% w/v sodium chloride, then concentrated under reduced pressure at jacket temperature ≤35 °C. Residues from incomplete bicarbonate washing can carry residual dicyclohexylurea into the crystallized product, causing a melting point depression of 2–4 °C relative to reference material.
Crystallization of CBZ-DL-phenylalanine from ethyl acetate/n-heptane mixtures is influenced by seed particle size distribution and cooling rate. In a 500 L crystallizer fitted with a retreat-curve impeller, controlled cooling from 45 °C to 5 °C at 0.2 °C/min yields a median particle diameter in the range of 80–120 μm; faster cooling increases the fine fraction below 20 μm and reduces filter throughput. Differential scanning calorimetry of commercial lots shows a broad melting endotherm that is polymorph-dependent; the specific onset and heat of fusion should be confirmed against the supplier certificate of analysis. Bulk density of the dried material typically lies between 0.45 g/mL and 0.60 g/mL after vacuum drying.
Removal of the carbobenzoxy group from CBZ-DL-phenylalanine on a production scale uses hydrogenation in a stirred autoclave equipped with a catalyst basket and gas-entrainment impeller. A representative charge loads CBZ-DL-phenylalanine at 0.5–1.0 mol/L in methanol or ethyl acetate with 5–10% w/w palladium on activated carbon (10% Pd/C) relative to substrate. The vessel is inerted with nitrogen, then pressurized with hydrogen to 1–4 bar at 20–40 °C. Carbon dioxide evolution during hydrogenolysis increases vessel pressure by 0.1–0.3 bar per mole of substrate depending on headspace volume; pressure release through a vent condenser is required to prevent exceeding the relief set point. Reaction endpoint is determined by HPLC disappearance of the starting material below 0.10% area. The catalyst is retained by a 5 μm sintered metal filter; catalyst breakthrough into the filtrate produces black specks in the isolated phenylalanine derivative and can inhibit the subsequent coupling step. Spent catalyst retains palladium and is segregated for precious-metal recovery. The deprotected species, DL-phenylalanine, is isolated as the hydrochloride or free amino acid depending on the downstream step.
Stability boundaries for CBZ-DL-phenylalanine are set by moisture uptake, thermal exposure, and incompatible process chemicals. Bulk material stored at 25 °C and relative humidity >60% shows measurable water uptake and caking within 48 h; storage under nitrogen at 2–8 °C in a sealed container is therefore specified for long-term use. The product is incompatible with strong acids such as hydrogen bromide in acetic acid and with strong oxidizers. Pre-drying at 60 °C under vacuum for 4 h is recommended when Karl Fischer titration exceeds 0.50% water. The material should not be micronized in unhumidified air streams because static charge accumulation on fine particles can exceed the ignition energy of organic dust; conductive grounding and inert-gas blanketing are specified in milling operations. The product also should be segregated from hydrogenation catalysts in storage and from reducing metal powders to avoid unintended deprotection in the dry state.
Field experience with bulk packaging indicates that fiber drums without an aluminum foil barrier can allow moisture ingress during repeated opening in tropical climates, leading to caking and a loss on drying above 0.50%. Transfer to conductive plastic intermediate bulk containers with nitrogen overlay is recommended for quantities above 100 kg. Batch-to-batch variation in particle size can alter dissolution time in coupling solvents; material with a median particle size above 150 μm may require extended stirring for complete dissolution in ethyl acetate at 20 °C.
