| HS Code | 231223 |
| Cas Number | 17609-53-7 |
| Molecular Formula | C16H15NO4 |
| Chemical Name | (2R)-2-(Benzyloxycarbonylamino)-2-phenylacetic acid |
| Synonyms | Z-D-Phenylglycine; N-Cbz-D-phenylglycine; Z-D-Phg-OH |
| Appearance | White to off-white powder |
| Melting Point | 124-128 °C |
| Optical Rotation | [α]D20 -115° (c=1, ethanol) |
| Solubility | Soluble in ethanol, methanol, DMF, and DMSO; sparingly soluble in water |
| Purity | ≥98% (HPLC) |
| Storage Conditions | Store at -20°C, dry, sealed, protected from light |
| Smiles | O=C(O)[C@@H](NC(=O)OCc1ccccc1)c2ccccc2 |
As an accredited CBZ-D-phenylglycine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | CBZ-D-phenylglycine (25 g) is packaged as a white crystalline powder in a sealed glass bottle, protected from light and moisture. |
| Container Loading (20′ FCL) | A 20′ FCL container safely loads CBZ-D-phenylglycine in sealed drums, palletized, secured, and ventilated for transport. |
| Shipping | CBZ-D-phenylglycine should be shipped at ambient temperature in a sealed, light-resistant container, protected from moisture. Handle as a non-hazardous chemical unless specified otherwise, but ensure compliance with local transport regulations. Include documentation with product name, purity, and safety data sheet for traceability and safe handling. |
| Storage | Store CBZ-D-phenylglycine in a tightly sealed container in a cool, dry, well-ventilated area. Protect from moisture, direct sunlight, and heat. Keep away from strong oxidizing agents and incompatible materials. Ensure the container is clearly labeled and stored at controlled room temperature to maintain stability and purity. |
| Shelf Life | Store tightly sealed in a cool, dry place; typical shelf life is 2–3 years under recommended conditions. |
Across solution-phase peptide contract manufacturing campaigns, CBZ-D-phenylglycine is pre-dissolved in anhydrous dimethylformamide dried to ≤0.02 wt% water by Karl Fischer titration per ASTM E203, then activated with 1.05–1.15 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in the presence of 1.0–1.1 equivalents of 1-hydroxybenzotriazole at 0–5°C. The free carboxyl group is converted to the HOBt ester, while the benzyloxycarbonyl group on the D-phenylglycine nitrogen remains intact. A tertiary base such as N-methylmorpholine is added to maintain an apparent pH of 5.5–6.5 during coupling to amine nucleophiles; below 5.0 the activation slows, and above 6.5 the risk of 5(4H)-oxazolone formation from the activated carboxyl increases. Racemization at the D-Phg α-carbon is quantified after total deprotection and derivatization by chiral HPLC on an immobilized amylose tris(3,5-dimethylphenylcarbamate) column with 254 nm detection; routine batch release limits for the L-Phe-D-Phg epimer are set at ≤0.5% relative area. Glass-lined stirred reactors are specified because residual iron ions from stainless steel vessels bind to HOBt and produce a reddish complex that lowers activation reproducibility. Coupling conversion is tracked by inline FT-IR for the disappearance of the carboxylic acid carbonyl at 1710 cm⁻¹; incomplete conversion below 95% after 6 h at 20–25°C triggers a supplementary addition of 0.15 equivalents of EDC·HCl rather than an increase in temperature. The workup includes a 5% citric acid quench to remove unreacted base and diisopropylurea by-products, followed by extraction into ethyl acetate. Diisopropylurea content in the isolated intermediate is controlled at ≤0.2% by gas chromatography with flame ionization detection.
In multikilo catalytic hydrogenation of CBZ-D-phenylglycine-containing peptide intermediates, the rated capacity of a hydrogenation autoclave is frequently set not by substrate mass but by carbon dioxide off-gas from the benzyloxycarbonyl group cleavage. Each mole of CBZ group liberates one mole of carbon dioxide and one mole of toluene upon hydrogenolysis; the gas-liquid mass transfer coefficient for hydrogen is reduced when the solvent is saturated with CO₂. A stirred autoclave equipped with a gas-entrainment impeller and baffle is operated at 1–4 bar hydrogen overpressure and 20–30°C. Temperature above 40°C promotes hydrogenolysis of the phenylglycine benzylic C–N bond and produces phenylacetic acid derivatives; chiral HPLC then shows an enantiomeric purity loss greater than 1%. Palladium on carbon at 5–10 wt% loading relative to substrate is poisoned by sulfur-containing peptide residues, methionine or cysteine; the observed turnover frequency can fall by more than 50%, requiring catalyst recharge and lengthening the batch cycle. The reaction solvent is held at pH 4.5–6.5 with acetic acid or ammonium formate because alkaline conditions saponify methyl and ethyl ester protecting groups elsewhere in the peptide. After reaction, the slurry is filtered through a 0.2 μm PTFE membrane together with a Celite bed; residual palladium in the isolated D-phenylglycine peptide is measured by ICP-MS according to ICH Q3D and must comply with the parenteral permitted daily exposure for palladium of 10 μg/day. Catalytic hydrogenolysis is incompatible with benzyl ether-protected serine or threonine residues, because those groups are also cleaved under the same conditions; in such sequences hydrogenolysis is replaced by acidolytic cleavage with HBr in acetic acid.
Release testing for CBZ-D-phenylglycine used in GMP peptide API manufacturing is carried out before the material is brought into classified production areas, because once the container is opened the powder can absorb moisture above 0.5 wt% and alter the mass balance in subsequent activation stages. A representative batch is sampled under dry nitrogen and analyzed by the compendial methods listed below. Chiral purity is not inferred from a single optical rotation value because trace amounts of the L-enantiomer can be masked by solvent impurities; orthogonal IR identification and chiral chromatographic retention time are required.
| Parameter | Method/Standard | Typical acceptance criterion |
|---|---|---|
| Identification | USP <197> | Infrared spectrum matches reference standard |
| Assay on dried basis | USP <621> HPLC | 98.0–102.0% |
| Chiral purity | Ph. Eur. 2.2.29 HPLC, chiral stationary phase | L-isomer ≤0.1% |
| Residual solvents | USP <467> headspace GC | Class 2 solvent limits per ICH Q3C |
| Loss on drying | USP <731> | ≤0.5% |
| Residue on ignition | USP <281> | ≤0.1% |
Acidolytic removal of the CBZ group in D-phenylglycine-containing peptides is performed with 30–33 wt% hydrogen bromide in glacial acetic acid at 0–20°C for 2–4 h. The liberated benzyl cation is scavenged by adding 10–15 equivalents of anisole or thioanisole; without a scavenger the benzyl cation alkylates electron-rich aromatic side chains such as tyrosine and tryptophan, producing benzyl-modified impurities that are detectable by LC-MS as +90 Da adducts. This method is selected when the larger peptide intermediate contains benzyl ether groups that must be preserved, provided those groups are stable to strong acid. The cleavage vessel must be glass-lined or PTFE-lined because HBr/acetic acid corrodes stainless steel and releases iron ions that bind to the liberated amino group. After cleavage, the product is precipitated from diethyl ether and the residual acetic acid is removed by repeated heptane trituration. Residual bromide in the final D-phenylglycine peptide is controlled by ion chromatography with a limit of ≤0.05% for parenteral applications. The main disadvantages are racemization of sensitive peptide bonds and partial hydrolysis of tert-butyl ester groups; the method is therefore not used when aspartic acid or glutamic acid side-chain tert-butyl protection is present. Catalytic transfer hydrogenation with ammonium formate is sometimes evaluated as an alternative, but published data for this specific configuration in peptide substrates is limited and each sequence requires screening.
CBZ-D-phenylglycine is converted to D-phenylglycinol for manufacture of C₂-symmetric bisoxazoline ligands used in asymmetric catalysis. The carboxylic acid is first activated as a mixed anhydride with isobutyl chloroformate and N-methylmorpholine in tetrahydrofuran at −20°C, then reduced with 1.2–1.5 equivalents of sodium borohydride in water or with borane-dimethyl sulfide at 0–5°C. Direct borane reduction without preactivation is slower and leads to cyclic urethane by-products under prolonged reaction times. The CBZ group remains intact during reduction; loss of the protective group at this stage produces free D-phenylglycinol that is water-soluble and difficult to extract, reducing isolated yield below 60%. After aqueous quench with 2 N hydrochloric acid, the product is extracted into methyl tert-butyl ether and isolated as the hydrochloride salt from ethanol/MTBE. Optical rotation is measured at 589 nm in methanol; a decrease greater than 1% relative to the input enantiomeric purity indicates racemization during the mixed anhydride step. The resulting CBZ-D-phenylglycinol is then deprotected by catalytic hydrogenolysis and transformed into the bisoxazoline ligand via a two-fold condensation with diethyl malonate derivatives. The stereochemical integrity of the ligand is confirmed by chiral HPLC on an immobilized cellulose tris(3,5-dimethylphenylcarbamate) column with hexane/ethanol mobile phase.
Semisynthetic β-lactam process development evaluates CBZ-D-phenylglycine as a crystalline protected side-chain donor when the Dane salt route is not available or when a solid, storable intermediate is required for pilot-plant scheduling. The carboxylic acid is activated in acetone/water at −10 to 0°C with methyl chloroformate and N-methylmorpholine, or with pivaloyl chloride, and then condensed with 6-APA or 7-ADCA nuclei. The mixed anhydride must be used within 10–15 min because decomposition regenerates protected D-phenylglycine and reduces the conversion. The coupled intermediate is subjected to hydrogenolysis in neutral aqueous tetrahydrofuran to remove the CBZ group; the mild neutral conditions preserve the β-lactam ring, whereas strongly acidic deprotection such as HCl in dioxane opens the β-lactam and generates the corresponding penicilloic or cephalosporoic acid. Critical process parameters are monitored by HPLC according to Ph. Eur. 2.2.29; known β-lactam degradation products are held below 0.1% area normalization. Published data for this specific CBZ-protected configuration in commercial β-lactam manufacturing is limited; most large-scale routes use the enamine or Dane salt protection because it avoids the separate hydrogenolysis step. The value of CBZ-D-phenylglycine in this sector is therefore mainly in process development, impurity-spiking studies, and preparation of reference batches, rather than as a direct replacement for established side-chain donors.
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CBZ-D-phenylglycine (CAS 17609-48-2, molecular formula C16H15NO4, relative molecular mass 285.29 g/mol) is the carboxybenzyl-protected D-enantiomer of α-phenylglycine, also designated Z-D-Phg-OH, N-benzyloxycarbonyl-D-2-phenylglycine, or N-Cbz-D-phenylglycine. The compound is released as a white to off-white crystalline powder for use as a chiral building block in solution-phase peptide synthesis and in the preparation of semisynthetic β-lactam intermediates. In peptide-synthesis grade material, the assay is typically controlled to not less than 98.0% by HPLC and the enantiomeric purity to not less than 99.0%; the principal stereochemical impurity is Cbz-L-phenylglycine, which co-migrates with the D-form under non-chiral reverse-phase conditions and is therefore quantified by chiral HPLC. The carboxybenzyl group masks the α-amino function as a benzyl carbamate, leaving the carboxylic acid free for conversion to the acid chloride, N-hydroxysuccinimide ester, pentafluorophenyl ester, or in situ carbodiimide-mediated activation.
Industrial packaging for this intermediate is usually supplied as 1 kg, 5 kg, and 25 kg net quantities in low-density polyethylene liners inside sealed fibre drums. The product is stored at 2–8 °C in a dry environment; storage under nitrogen is specified where prolonged retention of low moisture and colour stability are required. Material opened at relative humidity above 60% may adsorb water sufficiently to alter stoichiometry during acid chloride generation or carbodiimide coupling. Pre-drying under vacuum at 40 °C for 12–24 h is therefore applied before anhydrous activation when Karl Fischer water content exceeds 0.5%.
For release and stability evaluation, the following analytical matrix is applied. The acceptance limits shown are representative of peptide-synthesis grade material; supplier-specific limits may differ and should be verified against the certificate of analysis.
| Parameter | Method | Acceptance Criterion |
|---|---|---|
| Appearance | Visual inspection | White to off-white crystalline powder |
| Assay | HPLC, USP <621> | ≥98.0% peak area |
| Enantiomeric purity | Chiral HPLC, USP <621> | Cbz-D-phenylglycine ≥99.0%; Cbz-L-phenylglycine ≤1.0% |
| Water content | Karl Fischer, USP <921> | ≤0.5% |
| Residue on ignition | USP <281> | ≤0.1% |
| Specific optical rotation | Ph. Eur. 2.2.31 | Report value in methanol at 20 °C against qualified reference standard |
| Residual solvents | Ph. Eur. 2.4.24 | Complies with ICH Q3C Class 3 limits |
For chiral HPLC release testing, method qualification should establish resolution between Cbz-D-phenylglycine and Cbz-L-phenylglycine of not less than 1.5. Standard solutions are prepared in methanol or acetonitrile at 1.0 mg/mL; injection volume, column temperature, and detector wavelength are validated under USP <621>. Because the D- and L-forms possess nearly identical solubility, thermal properties, and retention on non-chiral stationary phases, chiral HPLC is mandatory for release. Where the material is intended for registered pharmaceutical intermediates, the residual solvent profile is governed by ICH Q3C. Trace metal data under ICH Q3D are less commonly disclosed for this product as a raw material; when downstream hydrogenolysis is used, palladium concentration in the isolated intermediate is verified by a suitable USP <233> inductively coupled plasma procedure. Published data for full elemental impurity profiles of Cbz-D-phenylglycine as a raw material are limited, and a dedicated risk assessment is applied when the material enters a route that includes a registered final drug substance.
The methine proton at the chiral centre of α-phenylglycine is rendered more acidic by the adjacent phenyl ring, and unprotected D-phenylglycine is susceptible to base-catalysed deprotonation and subsequent racemization through an oxazolone intermediate when the carboxylic acid is activated. The carboxybenzyl group does not eliminate this intrinsic C–H acidity; it protects the α-amino group from premature acylation and modifies the reactivity of the protected monomer. The decisive advantage of Cbz protection is process-dependent: the group is removed by hydrogenolysis over 5% palladium on carbon under 0.1–0.5 MPa hydrogen in methanol or tetrahydrofuran at 20–35 °C, or by hydrogen bromide in acetic acid. These conditions avoid the strongly basic secondary amine environment of Fmoc removal, which for α-phenylglycine derivatives is associated with increased enantiomeric erosion when piperidine exposure is prolonged. Cbz-D-phenylglycine is therefore preferred in solution-phase routes where the target stereocentre must survive multiple coupling and deprotection operations.
Cbz-D-phenylglycine is stable to trifluoroacetic acid under standard Boc removal conditions, enabling orthogonal protection of additional amine sites in the same sequence. It is also stable to piperidine in DMF, so it may be carried through a synthesis while Fmoc groups are removed elsewhere. The carboxybenzyl group is not compatible with hydrogenolysis-sensitive substrates, including those bearing unprotected benzyl esters, unsaturated olefins, or certain sulphide-containing residues; in such cases alternative protecting groups or non-hydrogenolytic cleavage conditions are required.
The selection among Cbz-, Fmoc-, and Boc-protected D-phenylglycine is not determined solely by cost or availability; it follows the global protecting-group strategy and the deprotection chemistry that downstream steps can tolerate. The principal process differences are summarised below.
| Protected form | Predominant deprotection reagent | Typical conditions | Stability to other deprotection systems |
|---|---|---|---|
| Cbz-D-phenylglycine | Hydrogen/palladium; HBr/acetic acid | 0.1–0.5 MPa H₂, 20–35 °C | Stable to piperidine/DMF; stable to dilute TFA |
| Fmoc-D-phenylglycine | Piperidine/DMF or 4-methylpiperidine/DMF | 20–25 °C, 5–20 min | Stable to TFA; unstable to secondary amines |
| Boc-D-phenylglycine | Trifluoroacetic acid/dichloromethane | 0–25 °C, 30–120 min | Stable to piperidine/DMF; stable to hydrogenolysis |
Fmoc-D-phenylglycine is directly compatible with routine Fmoc-solid-phase peptide synthesis, but repeated exposure to piperidine or 4-methylpiperidine in DMF at 20–25 °C can generate unacceptable enantiomeric excess loss for α-phenylglycine residues if deprotection times are not tightly controlled. Boc-D-phenylglycine is cleaved by trifluoroacetic acid in dichloromethane at 0–25 °C; this is useful when the route contains hydrogenolysis-sensitive groups, but the acidic cleavage is less selective when acid-labile side-chain protections are already present. Cbz-D-phenylglycine occupies a distinct position in industrial routes: it is base-stable, stable to dilute TFA, and removable under hydrogenolysis at relatively low pressure, but it requires catalytic hydrogenation equipment and subsequent palladium-removal steps in final isolation.
Cbz-L-phenylglycine is the enantiomer and is not interchangeable with Cbz-D-phenylglycine in β-lactam side-chain construction because the D-configuration is required for the biological activity of the resulting penicillins and cephalosporins. The specification for Cbz-L-phenylglycine as an impurity is commonly set at ≤1.0%, with stricter limits of ≤0.5% available for custom high-purity grades.
Conversion of Cbz-D-phenylglycine to the corresponding acid chloride is performed with phosphorus pentachloride in dichloromethane or with oxalyl chloride and catalytic DMF at temperatures between -10 °C and 0 °C. The reaction is strongly exothermic; in glass-lined pilot reactors, the chlorinating agent is added under jacket control so that the batch temperature remains below 5 °C until the evolution of hydrogen chloride and carbon monoxide has ceased. Residual chlorinating agent is removed by distillation or solvent displacement before the acid chloride is introduced into the coupling vessel.
The activated intermediate is coupled with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid in aqueous acetone or dichloromethane in the presence of inorganic base. The protected amide intermediate is then deprotected by catalytic hydrogenolysis to remove the carboxybenzyl group. This route is used in the preparation of ampicillin and cephalexin intermediates; the D-phenylglycine side chain is the structural determinant of the antibacterial spectrum of these β-lactams. Process yield and optical purity are monitored by HPLC at the protected intermediate stage and after deprotection, with the final D-phenylglycine content confirmed by chiral HPLC.
Direct coupling of Cbz-D-phenylglycine in solution-phase peptide synthesis is accomplished by conversion to the N-hydroxysuccinimide ester or by in situ activation with N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole in dry DMF. The free carboxylic acid has low aqueous solubility unless one equivalent of sodium bicarbonate is added to form the sodium carboxylate; this salt can be used for aqueous workup but is re-acidified before organic extraction and activation. Batches with water content above 0.5% often show reduced activation efficiency because the carbodiimide is hydrolysed by residual water, leading to N-acylurea byproducts and lower coupling yield.
After hydrogenolysis, palladium is removed by filtration through a pad of diatomaceous earth or a 0.45 µm membrane. Incomplete removal of palladium can lead to metallic contamination in the isolated D-phenylglycine intermediate; the filtrate is therefore tested for palladium by USP <233> before the free amino acid is converted to the final β-lactam salt. Filtration pressure should not exceed 0.2 MPa when using disposable membrane cartridges, as higher differential pressure may rupture the filter and release catalyst fines into the product stream.
Incompatibilities include strong oxidising agents, which can degrade the benzyl carbamate, and secondary amines in hot solution, which may slowly remove the Cbz group or promote racemization under forcing conditions. The material is not recommended for processes requiring anhydrous basic media above 40 °C without prior stability verification, because benzyl carbamate groups can undergo thermal decomposition with loss of carbon dioxide and benzyl alcohol.
