| HS Code | 127596 |
| Product Name | CBZ-L-Proline |
| Cas Number | 1148-11-4 |
| Molecular Formula | C13H15NO4 |
| Molecular Weight | 249.26 g/mol |
| Appearance | White to off-white crystalline powder |
| Melting Point | 75-78°C |
| Specific Optical Rotation | -60° (c=1, ethanol) |
| Purity | ≥98% |
| Solubility | Soluble in ethanol, methanol, DMSO, and DMF; sparingly soluble in water |
| Storage Conditions | Store at 2-8°C, protected from moisture |
| Chemical Class | N-protected amino acid |
| Mdl Number | MFCD00037317 |
As an accredited CBZ-L-Proline factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | CBZ-L-Proline is packaged in 100 g amber glass bottles with airtight seals, desiccant, and tamper-evident caps. |
| Container Loading (20′ FCL) | 20′ FCL container loading of CBZ-L-Proline: packed in sealed drums on pallets, secured, labeled, moisture-protected, with proper ventilation. |
| Shipping | CBZ-L-Proline should ship at ambient temperature in a sealed, light-resistant container, protected from moisture and extreme heat. Use sturdy outer packaging with absorbent material to prevent leakage. Ensure compliance with hazardous goods regulations if applicable. Avoid prolonged exposure to sunlight during transit. |
| Storage | Store CBZ-L-Proline in a tightly sealed container, protected from light and moisture, in a cool, dry, well-ventilated area at room temperature (2–8°C recommended for long-term stability). Avoid exposure to strong acids, bases, and oxidizing agents. Keep away from incompatible materials and ensure the container is clearly labeled. |
| Shelf Life | Shelf life is typically 2-3 years when stored dry, cool, and protected from light in a sealed container. |
In solution-phase peptide active pharmaceutical ingredient manufacture, Cbz-L-proline is charged as an N-protected proline building block that acts as a carboxyl donor in mixed anhydride-mediated segment condensation. The unprotected L-proline zwitterion is intentionally avoided because the free secondary amine can participate in intramolecular cyclization to diketopiperazine during carboxyl activation and because solubility in chlorinated or ethereal solvents is insufficient for the water-sensitive coupling sequence. A production-scale reaction train consisting of a jacketed glass-lined reactor, a metering pump for the mixed anhydride, and a hydrogenation autoclave is used. Cbz-L-proline is dissolved in dichloromethane or tetrahydrofuran at 0.8–1.2 mol/L, cooled to −15 to −5 °C, and neutralized with N-methylmorpholine at 1.05–1.10 mol per mol of Cbz-L-proline. Activation is carried out with isobutyl chloroformate or pivaloyl chloride at 0.98–1.03 mol per mol of Cbz-L-proline, with internal temperature controlled by jacket flow and measured by thermocouple. The mixed anhydride is subsequently added to a free-based amino acid ester solution; the charge ratio is maintained between 1.02 and 1.10 mol Cbz-L-proline per 1.00 mol amino ester to suppress residual free amine. After 30–60 min at 0–5 °C, the reaction is quenched with aqueous sodium bicarbonate, and the organic phase is washed until residual Cbz-L-proline measures below 0.15 area% by HPLC at 210 nm. The Cbz-protected peptide intermediate is then subjected to hydrogenolysis in an ethanol/water mixture using 5% Pd/C at 0.5–1.0 wt% on dry substrate basis under 0.3–0.5 MPa hydrogen pressure at 25–35 °C. The catalyst is removed by filtration through a 0.45 µm membrane, and the filtrate is concentrated for crystallization. Compliance for this intermediate stage is governed by ICH Q7, EU GMP Part II, and applicable pharmaceutical legislation; residual palladium is controlled by ICH Q3D and USP ⟨232⟩, residual solvents by USP ⟨467⟩, and chiral purity by monograph-validated chiral HPLC. Published data for this specific production configuration is limited; the ranges below represent equipment-specific control banding used in process qualification and are not universal formulation limits. Terminal product types are N-terminal proline-containing synthetic peptide drug substances and their protected intermediates, used in downstream formulation as injectable or lyophilized peptide APIs.
Representative in-process control ranges for the mixed anhydride coupling sequence:
| Control point | Range | Analytical basis |
|---|---|---|
| Activation temperature | −15 to −5 °C | Internal thermocouple / ICH Q7 batch record |
| Coupling charge ratio | 1.02–1.10 mol Cbz-L-proline per 1.00 mol amino ester | HPLC area% residual amine |
| Residual Cbz-L-proline before hydrogenolysis | <0.15 area% | HPLC 210 nm |
| Hydrogen pressure | 0.3–0.5 MPa | Pressure transmitter / USP ⟨467⟩ |
| Residual palladium | ≤10 µg/g oral PDE | ICP-MS / USP ⟨232⟩, ICH Q3D |
When Cbz-L-proline is converted to an N-hydroxysuccinimide or pentafluorophenyl ester before coupling, the pre-activation shifts the rate-limiting step from carboxyl activation to aminolysis and reduces base-mediated abstraction at the Cα position that would otherwise occur with in situ coupling reagents. Proline is less prone to oxazolone formation because the Cbz group blocks the nitrogen, but active ester formation must still be controlled with respect to water content and temperature. The protected amino acid is dissolved in anhydrous tetrahydrofuran or N,N-dimethylformamide at 0.8–1.5 mol/L; N-hydroxysuccinimide is charged at 1.10–1.20 mol per mol of Cbz-L-proline, and dicyclohexylcarbodiimide is charged at 1.05–1.15 mol per mol of Cbz-L-proline at 0–5 °C. The water content of the solvent is maintained below 0.05% by Karl Fischer titration to avoid active ester hydrolysis. After 12–18 h at 4–8 °C, the precipitated dicyclohexylurea is removed by filtration or centrifugation, and the active ester is isolated by non-aqueous precipitation. The isolated Cbz-L-proline active ester is then coupled in a separate reactor to a peptide segment bearing a single free amine; the active ester charge is 1.00–1.10 mol per mol of free amine, and the pH is adjusted with N-methylmorpholine or N,N-diisopropylethylamine to 8.0–8.5 in N,N-dimethylformamide. Reaction progress is monitored by HPLC, and chiral purity after workup is confirmed to be ≥99.0% enantiomeric excess. The process is conducted under ICH Q7 when the peptide segment is an API intermediate, with ISO 9001:2015 quality management for non-pharmaceutical custom synthesis and REACH (EC) No 1907/2006 for the European market. Terminal product types are N-terminal Cbz-proline-protected peptide segments, which are further deprotected and chain-extended to peptide APIs or fine chemical peptide intermediates.
The conversion of Cbz-L-proline to (S)-diphenylprolinol, a chiral amino alcohol used in oxazaborolidine-catalyzed enantioselective ketone reduction, begins with the reduction of the carboxylic acid to the primary alcohol. Cbz-L-proline is dissolved in tetrahydrofuran at 0.5–1.0 mol/L and reduced with lithium aluminium hydride at 1.2–1.5 mol per mol substrate under 0–10 °C; alternatively, a sodium borohydride–iodine system is used to reduce handling risk at 0–10 °C. The reaction is quenched with ethyl acetate and aqueous sodium hydroxide, and the resulting N-Cbz-L-prolinol is extracted and concentrated. The alcohol is then dissolved in anhydrous tetrahydrofuran and treated with phenylmagnesium bromide at 2.2–2.5 mol per mol alcohol at 0–25 °C in a jacketed reactor with controlled addition over 2–4 h. After aqueous ammonium chloride quench, solvent swap to isopropanol, and crystallization, the N-Cbz-diphenylprolinol intermediate is isolated. Hydrogenolysis over 5% Pd/C at 0.3–0.5 MPa hydrogen removes the Cbz group to yield (S)-diphenylprolinol, which is complexed with borane–tetrahydrofuran in anhydrous tetrahydrofuran to generate the oxazaborolidine catalyst. In the downstream ketone reduction, the oxazaborolidine catalyst is charged at 1–10 mol% relative to the prochiral ketone, typically 5 mol% when the ketone is substituted with an electron-withdrawing group; the ketone is reduced in tetrahydrofuran at 0–25 °C, and the product chiral alcohol is isolated by extraction and distillation or crystallization. Compliance for the catalyst manufacturing stage is governed by ISO 9001:2015 and REACH (EC) No 1907/2006; if the catalyst is used in a registered pharmaceutical route, the sourcing and quality controls follow ICH Q7, and residual palladium is monitored by USP ⟨232⟩ or equivalent. Terminal product types are chiral alcohol building blocks used in subsequent pharmaceutical and fine chemical synthesis, as well as the oxazaborolidine–borane complex itself as a non-isolated or isolated process reagent.
In the manufacture of proline-derived organocatalysts for asymmetric aldol and Michael additions, Cbz-L-proline functions as a masked secondary amine that protects the pyrrolidine nitrogen during carboxyl derivatization to amides or esters. The protected amino acid is coupled with an amine donor such as pyrrolidine or aniline using a carbodiimide-mediated activation in dichloromethane at 0–5 °C, with the amine charged at 1.00–1.10 mol per mol of Cbz-L-proline and the carbodiimide at 1.05–1.15 mol per mol of Cbz-L-proline. After 8–12 h, the dicyclohexylurea by-product is filtered, and the Cbz-protected amide is hydrogenolysed over 5% Pd/C in ethanol at 0.3–0.5 MPa hydrogen to release the pyrrolidine nitrogen. The resulting secondary amine catalyst is isolated as a free base or hydrochloride salt and then used in asymmetric aldol reactions at a catalyst loading of 5–20 mol% relative to the aldehyde donor, with acetone or another ketone donor charged in large excess at 5.0–10.0 mol per mol aldehyde. The reaction is conducted in dimethyl sulfoxide or N,N-dimethylformamide at 20–25 °C for 24–72 h, followed by extractive workup and chiral HPLC analysis. Compliance for this downstream segment is typically governed by ISO 9001:2015, REACH (EC) No 1907/2006, and, when the chiral product enters a pharmaceutical route, ICH Q7 analytical controls; residual solvents are controlled by USP ⟨467⟩. Terminal product types are chiral β-hydroxy ketone intermediates and related asymmetric aldol adducts used in fine chemical and pharmaceutical intermediate synthesis. Published process data for this specific configuration is limited; the catalyst loading range is determined experimentally from enantioselectivity data and is not a formulation specification for a finished drug product.
During contract research and process development for synthetic peptide APIs, Cbz-L-proline is used as a solution-phase building block in route scouting because the Cbz group provides orthogonal protection that can be removed independently from methyl, tert-butyl, and benzyl ester groups, allowing sequential C- and N-terminal chain extension without a global deprotection step. Parallel route-scouting campaigns are run in jacketed laboratory reactors or multireactor synthesis workstations; the protected amino acid is charged at 1.05–1.20 mol per mol of the limiting peptide segment amine to assess coupling performance across solvent systems. Process conditions evaluated include activation with isobutyl chloroformate at −15 to −5 °C, coupling in tetrahydrofuran or dimethylformamide at 0–25 °C, and hydrogenolysis over 5% Pd/C at 0.2–0.4 MPa hydrogen. HPLC, LC-MS, and chiral HPLC are used to track residual starting material, diastereomer formation, and chiral purity. Compliance for route-scouting activities is governed by ICH Q11 for starting material justification when the route is transferred to registration, ICH Q7 for GMP manufacturing after process qualification, and ISO 9001:2015 for research quality management. Terminal product types from this downstream activity are qualified synthetic route definitions, demonstration batches of N-terminal proline-containing peptide intermediates, and technology transfer packages, rather than commercial finished dosage forms.
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Commercial material identified as CBZ-L-Proline, formally designated (2S)-1-[(benzyloxy)carbonyl]pyrrolidine-2-carboxylic acid and registered under CAS 1148-11-4, is an N-protected L-proline derivative used as a chiral intermediate in solution-phase peptide synthesis and as a starting material for proline-derived building blocks. The substance is also supplied under the synonyms Z-Pro-OH, Z-L-Pro-OH, and N-Cbz-L-proline. It has the molecular formula C13H15NO4, a formula weight of 249.26 g/mol, and a monoisotopic mass of 249.1001 Da. Standard packaging configurations include 25 g, 100 g, 500 g, and 1 kg units in amber glass or sealed polypropylene containers under inert gas; 5 kg and 25 kg fibre drums with polyethylene liners are used for pilot-scale process development. Research-grade material is typically released with assay by HPLC at ≥ 99.0% area and enantiomeric purity ≥ 99.5% area for the L-isomer. The product appears as a white to off-white crystalline powder with a capillary melting range of 76.0–79.0 °C measured according to USP <741>. Specific rotation is controlled within -58.0° to -62.0° at c = 1.0 in ethanol at 20 °C using the sodium D line at 589 nm. The material dissolves in methanol, ethanol, ethyl acetate, dichloromethane, tetrahydrofuran, and N,N-dimethylformamide; aqueous solubility is pH-dependent because the free carboxylic acid forms water-soluble salts under mildly basic conditions.
In a representative solution-phase sequence, CBZ-L-Proline functions as a temporary N-terminal protecting group for the proline residue. The N-hydroxysuccinimide ester of CBZ-L-Proline can be prepared with N,N’-dicyclohexylcarbodiimide and isolated by filtration, then used to acylate amine-functionalized resins or solution-phase substrates at loading densities from 0.3 mmol/g to 1.0 mmol/g. The carbamate protection prevents uncontrolled acylation at the proline nitrogen while leaving the carboxyl group available for selective activation. Coupling to amine-functionalized resins is monitored by HPLC after cleavage of the Cbz group from the resin-bound material.
Release testing combines identification, assay, enantiomeric purity, water content, residual solvents, and elemental impurity screening. The reversed-phase HPLC assay uses a 250 mm × 4.6 mm, 5 μm C18 column with a mobile phase gradient of 0.1% trifluoroacetic acid in water and acetonitrile, ultraviolet detection at 210 nm. System suitability requires resolution of at least 1.5 between the L-isomer and the nearest process-related impurity. Enantiomeric purity is determined by normal-phase chiral HPLC on a 250 mm × 4.6 mm, 5 μm CHIRALPAK AD-H column with hexane/ethanol/trifluoroacetic acid mobile phase; the D-enantiomer is reported as area percent against the L-isomer.
| Parameter | Acceptance criterion | Method or equipment |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection under fluorescent light |
| Identification | IR spectrum matches reference standard | FTIR-ATR, 4000–400 cm⁻¹ |
| Assay | ≥ 99.0% area | RP-HPLC, C18, UV 210 nm |
| Enantiomeric purity | ≥ 99.5% area L-isomer | Chiral HPLC, CHIRALPAK AD-H |
| Specific rotation | -58.0° to -62.0° at c = 1.0, ethanol, 20 °C | Polarimeter, sodium D line, 589 nm |
| Melting range | 76.0–79.0 °C | USP <741> capillary method |
| Water content | ≤ 0.5% w/w | Karl Fischer coulometric titration, USP <921> Method Ic |
| Residual solvents | Ethanol ≤ 5000 ppm; ethyl acetate ≤ 5000 ppm | Headspace GC-FID, USP <467>, ICH Q3C Class 3 |
| Sulfated ash | ≤ 0.1% | USP <281> |
Because the Cbz group is introduced through reaction of L-proline with benzyl chloroformate under aqueous alkaline conditions, batch-specific impurity profiles frequently include residual benzyl alcohol, benzyl carbonate derivatives, and inorganic salts. Headspace GC-FID monitors benzyl alcohol at a reporting threshold below 0.1% area; excessive benzyl alcohol carryover into peptide coupling steps can compete with activated esters and reduce coupling rates. Residual sodium chloride from neutralized reaction liquor is not routinely detected by HPLC but is limited through aqueous extraction and recrystallization. Sulfated ash remains below 0.1%, and ion chromatography for chloride is applied when the material is used in water-sensitive organometallic steps.
CBZ-L-Proline is generally resistant to oxazolone-mediated racemization because N-alkoxycarbonyl protection removes the amide nitrogen’s ability to form the aromatic oxazolone intermediate that dominates in N-acyl amino acids. Nonetheless, carboxyl activation with carbodiimides at temperatures above 25 °C can increase anhydride formation and epimerization at C2. Standard coupling protocols therefore use 1.0–1.2 equivalents of CBZ-L-Proline relative to the amine component, 1.0–1.1 equivalents of N,N’-dicyclohexylcarbodiimide or N,N’-diisopropylcarbodiimide, and 1.0 equivalent of 1-hydroxybenzotriazole in anhydrous DMF or dichloromethane at 0–5 °C under nitrogen. Reaction progress is monitored by HPLC at 210–215 nm until residual CBZ-L-Proline is below 1.0% area. The temperature window is deliberately narrow because published data for epimerization of highly hindered amine substrates under carbodiimide activation is limited; chiral HPLC of the crude coupling product against a CBZ-D-proline-derived standard is used to establish acceptance limits before scale-up.
Free L-proline differs from CBZ-L-Proline in solubility and reactivity. The unprotected amino acid exists as a zwitterion in aqueous media and is poorly soluble in aprotic solvents; CBZ-L-Proline is compatible with anhydrous coupling conditions. Direct coupling of free L-proline without N-protection can lead to oligomerization and uncontrolled acylation at both the α-nitrogen and the carboxylic acid site. The Cbz-protected derivative therefore provides a single defined reactive centre at the carboxyl group after activation, which is essential for sequence-specific peptide assembly.
Three N-protected proline derivatives dominate process chemistry: CBZ-L-Proline, Boc-L-proline, and Fmoc-L-proline. The benzyloxycarbonyl group is removed by hydrogenolysis or strong Lewis acids, but remains intact under the acidic conditions used to remove Boc and the secondary-amine conditions used to remove Fmoc. This orthogonality allows CBZ-L-Proline to be used in solution-phase sequences where side-chain protecting groups or N-terminal groups require differential removal.
| Property | CBZ-L-Proline | Boc-L-Proline | Fmoc-L-Proline |
|---|---|---|---|
| CAS registry number | 1148-11-4 | 15761-39-4 | 71989-31-6 |
| Molecular formula | C13H15NO4 | C10H17NO4 | C20H19NO4 |
| Formula weight (g/mol) | 249.26 | 215.25 | 337.37 |
| Protecting group lability | Hydrogenolysis or strong acid; stable to piperidine and TFA/DCM | Acid-labile; stable to base and mild hydrogenolysis | Base-labile; stable to acid and mild hydrogenolysis |
| Typical deprotection | 10% Pd/C, H₂ at 1–4 bar, 20–40 °C, 2–24 h in THF/EtOAc | 20–50% TFA in DCM, 0–25 °C, 0.5–2 h | 20% piperidine in DMF, 5–20 min |
| Primary process limitation | Residual palladium removal and hydrogenator fouling | Volatile TFA handling and acid-sensitive substrate incompatibility | Dibenzofulvene by-product management |
In Fmoc-based solid-phase peptide synthesis, CBZ-L-Proline is not removed by the standard 20% piperidine in DMF cycle. Its use on resin therefore requires a separate hydrogenolysis step, which is less convenient and may require specialized pressure equipment. It is selected when a proline residue must remain protected through multiple piperidine deprotection cycles while other Fmoc groups are removed; the resin-bound Cbz-proline is then cleaved by catalytic transfer hydrogenation before final resin cleavage. The approach is constrained by diffusion limitations inside the resin pores, and published data for this specific configuration is limited; swelling measurements in DMF and dichloromethane are used to optimize solvent exchange before hydrogenolysis.
Hydrogenolytic removal of the benzyloxycarbonyl group from CBZ-L-Proline-containing intermediates is performed in a stirred pressure vessel at hydrogen pressure between 1 bar and 4 bar and temperature between 20 °C and 40 °C. Carbon-supported palladium catalysts, typically 10% Pd/C with 50% water wetting, are charged at 5–10 weight percent relative to substrate. Ethyl acetate, tetrahydrofuran, ethanol, or mixtures of ethyl acetate and tetrahydrofuran are used because they dissolve both the substrate and the toluene cleavage product while maintaining adequate hydrogen mass transfer. A pre-slurry of catalyst in solvent for 15 minutes before substrate addition minimizes localized exotherm. Reaction completion is confirmed by HPLC disappearance of the Cbz-protected intermediate and by formation of the free amine. Hydrogen uptake ceases at 1 molar equivalent of hydrogen per Cbz group; prolonged reaction after uptake stops increases the risk of further hydrogenation of aromatic species and can reduce catalyst selectivity. At temperatures above 50 °C or pressures above 5 bar, the probability of proline ring hydrogenolysis and aromatic ring hydrogenation increases. The workup includes catalyst removal through a 0.2 μm PTFE membrane or a bed of diatomaceous earth, followed by acidic or basic extraction to isolate the deprotected proline derivative.
Transfer hydrogenation provides an alternative when pressure-rated hydrogenation equipment is not available. Ammonium formate, cyclohexene, or 1,4-cyclohexadiene in the presence of Pd/C generates hydrogen in situ and removes the Cbz group under mild reflux. The method is convenient for laboratory-scale reactions but is less atom-economical at pilot scale due to the formation of formate decomposition by-products. When transfer hydrogenation is used, residual formate is detected by ion chromatography and removed by aqueous washing before the next coupling step.
During catalytic hydrogenolysis, the cleavage product is toluene, not benzyl alcohol; however, residual benzyl alcohol from incomplete manufacturing purification or carbamate hydrolysis can persist in the substrate and is monitored separately. Residual toluene is removed by evaporation or azeotropic distillation before the next step. Residual benzyl alcohol is quantified by GC-FID with an acceptance threshold below 0.1% area before proceeding to coupling, because benzyl alcohol can be acylated in downstream steps and produce benzyl ester impurities.
Deprotection with heterogeneous palladium can leave dissolved palladium in the product stream. For pharmaceutical intermediates, residual palladium is controlled according to ICH Q3D classification as a Class 2B elemental impurity. Quantification by ICP-MS after microwave-assisted acid digestion is used with an acceptance criterion typically not exceeding 10 ppm for oral drug substances; parenteral applications may require lower limits. Removal methods include adsorption on silica-thiol scavenger resin, treatment with activated carbon at 20–40 °C, or precipitation with dithiocarbamate; selection depends on the metal coordination properties of the downstream peptide. Scavenger resins are preferable when the substrate contains thioether or amine groups that can bind to activated carbon and reduce yield. The residual palladium level is re-analysed after 1 hour of scavenging at 20–25 °C; if the limit is not met, the treatment is repeated with fresh scavenger rather than extending contact time, because prolonged contact can increase product degradation.
Diketopiperazine formation is a process hazard when hydrogenolysis liberates the N-terminal amine of a dipeptide ester in the same vessel. The free amine can attack the C-terminal ester intramolecularly, forming a cyclic diketopiperazine with loss of alcohol. To suppress this side reaction, hydrogenolysis is typically performed with one equivalent of acetic acid or with temperature control at 20 °C, and the free amine is acylated immediately after filtration. Diketopiperazine formation is monitored by HPLC as the disappearance of the linear intermediate and the appearance of a cyclic by-product with retention time distinct from the parent dipeptide ester.
CBZ-L-Proline should be stored in a dry, light-protected environment at 2–8 °C or 20–25 °C with low relative humidity; the lower range is recommended for long-term storage exceeding 24 months. The product is hygroscopic enough that repeated opening at ambient relative humidity above 60% increases water content and can reduce coupling yield in water-sensitive reactions. Containers are sealed under nitrogen or argon after each withdrawal. Before use in peptide coupling, water content is re-checked by Karl Fischer titration; material with water content above 0.5% w/w is dried under vacuum at 40 °C to constant weight. Avoid contact with strong bases above pH 10 and prolonged exposure to temperatures above 60 °C, which can cause partial hydrolysis of the carbamate. Dust formation during charging is controlled with local exhaust ventilation, and operations involving bulk powder are conducted under inert conditions in a ventilated enclosure. Incompatibility is noted with strong reducing agents, acid chlorides, and strong oxidizing agents; these can cleave or modify the benzyloxycarbonyl group before the intended hydrogenolysis step.
