| HS Code | 662071 |
| Name | CBZ-L-Tryptophan |
| Synonyms | Z-Trp-OH; N-(Benzyloxycarbonyl)-L-tryptophan |
| Cas Number | 7432-21-9 |
| Molecular Formula | C19H18N2O4 |
| Appearance | White to off-white crystalline powder |
| Melting Point | 124-128 °C |
| Specific Rotation | -2.0° (c=1, ethanol) |
| Solubility | Soluble in DMSO, DMF, methanol, ethanol; slightly soluble in water |
| Storage Conditions | Store at 2-8 °C, protected from light |
| Purity | ≥98% |
| Density | 1.3 g/cm³ (estimated) |
As an accredited CBZ-L-Tryptophan factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 25 g of CBZ-L-Tryptophan supplied as a white crystalline powder in a sealed glass vial with tamper-evident cap and label. |
| Container Loading (20′ FCL) | Load CBZ-L-Tryptophan in sealed drums into 20' FCL, secure cargo, avoid moisture, heat, and contamination. |
| Shipping | CBZ-L-Tryptophan is typically shipped at ambient temperature in tightly sealed, moisture-resistant containers, protected from light and excessive heat. As a biochemical derivative, it is generally non-hazardous, but standard laboratory handling applies. Ensure packaging is intact, avoid prolonged exposure to humidity, and store under recommended conditions upon receipt. |
| Storage | Store CBZ-L-Tryptophan in a tightly sealed container, protected from light and moisture, ideally at -20°C. Keep away from oxidizers and strong acids/bases. Minimize repeated freeze-thaw cycles to preserve purity. Use dry, clean spatulas and allow the vial to warm to room temperature before opening. |
| Shelf Life | Shelf life: Store tightly sealed in a cool, dry place; typically stable for 2 years under recommended conditions. |
A high-volume downstream route for CBZ-L-tryptophan in GMP peptide active pharmaceutical ingredient manufacturing is solution-phase fragment condensation, where the N-benzyloxycarbonyl group is selected because it can be removed by catalytic hydrogenolysis rather than by repeated base exposure. In this process, the compound is stored at 2–8 °C and protected from relative humidity above 60% RH; if transfer must occur in humid conditions, nitrogen purging is used to limit carbodiimide hydrolysis during coupling. The coupling step uses 1.05–1.20 mol CBZ-L-tryptophan per 1.0 mol free amine component, with pre-activation at 0–5 °C in anhydrous DMF or DMF/THF using 1.05–1.10 mol DIC and 1.05–1.10 mol HOBt per 1.0 mol CBZ-L-tryptophan. The jacketed glass-lined reactor maintains ±2 °C control because activation above 8 °C accelerates oxazolone formation and subsequent α-carbon racemization. After coupling and aqueous workup, the Cbz group is removed in a stirred-tank hydrogenator over 5–10% Pd/C (50% water wet) under 1–4 bar hydrogen at 20–35 °C, with a hydrogen mass-flow controller and a 0.45 µm catalyst retention filter. The critical process conflict in this route is over-reduction of the indole side chain to the corresponding indoline impurity when hydrogen pressure exceeds 5 bar or temperature exceeds 40 °C; those impurities co-elute with the desired peptide in reversed-phase HPLC and are difficult to reject by crystallisation. Chlorinated solvents are excluded from the hydrogenation because palladium-catalysed dehalogenation consumes hydrogen and generates acid. Above 50 L working volume, baffled reactor geometry and bottom-outlet filtration become decisive because catalyst settling produces hot spots and incomplete deprotection if agitation is interrupted. Compliance with ICH Q7 Chapters 7 and 8 covers material quarantine and production operations, while 21 CFR 211.84 governs component testing and release, and USP <621> HPLC is used for enantiomeric purity and residual Cbz quantification. Terminal finished products are lyophilised acetate salts of therapeutic peptide APIs in the 5–25 amino acid residue range. Residual Cbz is established as a critical quality attribute during process validation rather than through a universal monograph limit; validated processes commonly report release thresholds at or below 0.10% peak area.
Cosmetic peptide active ingredient synthesis uses the same Cbz/indole chemistry but operates under a different regulatory logic: the final peptide is a cosmetic ingredient subject to Regulation (EC) No 1223/2009, while the raw-material synthesis is commonly controlled under EFfCI GMP 2017 and ISO 9001:2015 rather than pharmaceutical GMP. In this application, CBZ-L-tryptophan is coupled at 1.05–1.10 mol per 1.0 mol of the growing peptide fragment in anhydrous tetrahydrofuran at 0–5 °C using mixed-anhydride activation with isobutyl chloroformate and N-methylmorpholine; the lower coupling ratio relative to solid-phase excess protocols is selected because cosmetic-grade short peptides are built by stepwise solution-phase assembly in 2–4 residues. The Cbz group is then removed by mild hydrogenolysis over 5% Pd/C at 1–2 bar hydrogen pressure and 20–25 °C, followed by filtration through a 0.45 µm membrane and vacuum distillation to remove solvent. The terminal finished products are short-chain peptide actives supplied as lyophilised or spray-dried powders for emulsion-based anti-aging and barrier-repair formulations. In such materials, residual palladium and residual solvents are controlled to the sensitivity requirements of the leave-on cosmetic matrix, and published batch data for Cbz-derived peptide impurities in final formulations remain limited, so each downstream formulator must validate trace impurity compatibility in the allocated oil-water emulsion system. The hydrogenation step should not be operated in chlorinated solvents because reductive dehalogenation consumes hydrogen and can acidify the process stream, causing cosmetic peptide degradation before isolation.
Custom synthesis laboratories supplying early drug discovery programs use CBZ-L-tryptophan in small-batch solution-phase fragment coupling at 1.05 mmol per 1.0 mmol free amine, with DIC/HOBt activation in DMF at ambient temperature, followed by Cbz hydrogenolysis over 10% Pd/C under balloon pressure and C18 preparative HPLC; this route is confined to 0.1–10 mmol scale because catalyst settling and filtration dead volumes above 100 mmol require revalidation of mass transfer, and the terminal products are research-grade lyophilised peptide fragments supplied with certificates of analysis under ISO 9001:2015, with analytical release data aligned to ISO/IEC 17025:2017 for chromatographic purity and residual solvent determination.
The preparation of chromogenic protease substrates from CBZ-L-tryptophan proceeds directly from the carboxylic acid, with the N-benzyloxycarbonyl group retained in the final substrate to provide the hydrophobic N-terminal recognition element for serine protease active sites. In a representative mixed-anhydride activation, 1.0 mol CBZ-L-tryptophan is dissolved in anhydrous tetrahydrofuran at −15 to −10 °C, treated with 1.1–1.3 mol isobutyl chloroformate and 1.1–1.3 mol N-methylmorpholine, and then condensed with 1.0–1.1 mol p-nitroaniline or 2-naphthylamine. The downstream process includes acidic quench, ethyl acetate extraction, crystallisation from aqueous ethanol, and final vacuum drying; no hydrogenolysis is used because the Cbz group is part of the substrate structure. If the intermediate is intended for in vitro diagnostic kit incorporation, it is manufactured under ISO 13485:2016; if it is exported as a research-use-only enzyme substrate, ISO 9001:2015 and REACH Regulation (EC) No 1907/2006 Annex VI apply. The terminal products are chromogenic substrates such as CBZ-L-tryptophan p-nitroanilide, used in chymotrypsin and tryptophan aminopeptidase activity assays. The main stability constraint is the lability of the p-nitroanilide amide bond under strongly alkaline mobile phases, so storage and assay buffers must remain below pH 8.5 to avoid spontaneous hydrolysis. Aqueous workup under pH above 9.0 is incompatible with this substrate class because it accelerates cleavage of the chromogenic amide before the material can be isolated.
Where chiral tetrahydro-β-carboline scaffolds are required, CBZ-L-tryptophan serves as a crystalline protected precursor that is hydrogenolyzed to L-tryptophan immediately before a Pictet-Spengler cyclization with an aldehyde. The addition ratio after deprotection is 1.0–1.2 mol aldehyde per 1.0 mol L-tryptophan, with trifluoroacetic acid at 1–5 mol% as catalyst in dichloromethane at 20–25 °C; the Cbz group is removed in advance using 10% Pd/C and 1–2 bar hydrogen in methanol/water. The downstream production sequence includes catalyst filtration, solvent exchange into dichloromethane, aldehyde addition, and ageing for 12–24 h under nitrogen; stereochemical integrity is monitored by chiral HPLC because prolonged acid exposure beyond 24 h erodes enantiomeric excess at the newly formed stereocenter. This branch operates under ISO 9001:2015 and REACH (EC) No 1907/2006; terminal products are chiral tetrahydro-β-carboline hydrochloride salts and their N-Cbz protected intermediates for medicinal chemistry building block libraries. Published data for the exact aldehyde scope under these specific conditions is limited, so each new aldehyde substrate must be validated for cyclization rate and impurity profile before scale-up. Ketone-derived aldehydes containing enolisable centres require strict temperature control during condensation because competing aldol pathways consume aldehyde and generate non-crystalline by-products that complicate filtration.
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CBZ-L-tryptophan, also named N-[(benzyloxy)carbonyl]-L-tryptophan or N-Cbz-L-tryptophan, is a protected α-amino acid with the molecular formula C19H18N2O4 and molar mass 338.36 g mol⁻¹. The CAS registry number is 7432-21-5. The molecule retains the free indole side chain and the C-terminal carboxylic acid, while the α-amino group is blocked by a benzyl carbamate. This substitution pattern makes the compound suitable for solution-phase peptide synthesis, where selective deprotection by hydrogenolysis or acidolysis is required. The product is not defined by a universal model code; suppliers differentiate peptide-synthesis grade from custom fine-chemical grade by the completeness of analytical release data, particularly enantiomeric purity and residual-solvent documentation. Typical release criteria include chromatographic purity ≥98.0% and enantiomeric purity ≥99.0%. The material is supplied as a white to off-white powder and should be stored at 2–8 °C in tightly closed containers under inert gas. Because the free carboxylic acid can adsorb moisture, exposure to humid air should be limited. The solubility profile is pH-dependent: the compound is freely soluble in dimethylformamide and dimethyl sulfoxide, moderately soluble in methanol and ethyl acetate, and only sparingly soluble in neutral water, but dissolution improves above pH 8 as the carboxylate salt forms. Characterization by infrared spectroscopy typically shows overlapping carbonyl absorptions in the 1680–1740 cm⁻¹ region corresponding to the carbamate and carboxylic acid functions, although the exact band positions are not diagnostic alone and should be confirmed by 1H NMR or high-resolution mass spectrometry.
The Cbz function is removed by catalytic hydrogenolysis over palladium catalysts or by strong acid, most commonly hydrogen bromide in acetic acid. This profile contrasts with the Fmoc group, which is removed by secondary amines such as 20% piperidine in dimethylformamide through β-elimination, and with the Boc group, which is removed by acidolysis with trifluoroacetic acid. Because Cbz withstands the brief acid and base treatments used for Boc and Fmoc deprotection, it is used in convergent solution-phase syntheses where a tryptophan residue must remain protected while other protecting groups are removed. In practice, CBZ-L-tryptophan remains unchanged after exposure to 20% piperidine in dimethylformamide for 30 min at 20–25 °C, and it is generally retained under anhydrous trifluoroacetic acid treatment at 0–25 °C for short periods. Fmoc and Boc groups are generally stable under catalytic hydrogenation, so Cbz removal can proceed in their presence if no other reducible functionality is present. The orthogonality of Cbz therefore depends on the absence of competing hydrogenation-sensitive groups rather than on any single universal deprotection rule.
Because CBZ-L-tryptophan is not the subject of a dedicated pharmacopeial monograph in USP or Ph. Eur., the release data package is supplier-defined and typically derived from reversed-phase HPLC, chiral HPLC, headspace gas chromatography, and Karl Fischer coulometry. A representative specification matrix is shown in Table 1.
| Parameter | Method | Typical acceptance criterion |
|---|---|---|
| Appearance | Visual inspection | White to off-white powder |
| Assay | Reversed-phase HPLC, C18 5 µm, 250 × 4.6 mm, water/acetonitrile with 0.1% trifluoroacetic acid, UV 220 nm | ≥98.0% area |
| Enantiomeric purity | Chiral HPLC, amylose-based chiral stationary phase, hexane/2-propanol/trifluoroacetic acid | ≥99.0% area |
| Loss on drying | Karl Fischer coulometry | ≤0.5% |
| Residue on ignition | USP <281> | ≤0.2% |
| Heavy metals | ICP-MS | ≤10 ppm |
| Residual solvents | Headspace GC-FID | ICH Q3C class 3 ≤0.5% combined; class 2 within option 1 limits |
These values should be regarded as representative of a high-purity peptide-synthesis grade, not as a consequence of a single validated pharmacopeial procedure. When the product is used in GMP-regulated intermediate manufacture, the relevant certificate of analysis must include method-specific limits for the reaction solvents employed in the final crystallization; a generic “residual solvents comply” statement is insufficient for drug-substance supply chains. The 0.5% loss-on-drying threshold is practically relevant because residual moisture depresses the apparent assay and can affect anhydride formation during carbodiimide-mediated coupling. In a 2% dimethylformamide solution, the product should yield a clear to slightly opalescent liquid; persistent turbidity after 30 min of stirring at 20–25 °C may indicate particulate matter or incomplete solubilization.
In route selection, Cbz-L-tryptophan is specified when the final deprotection must not use piperidine or trifluoroacetic acid but can tolerate hydrogenation or acidolysis. A common scenario is the solution-phase assembly of a protected peptide acid: the Cbz group remains attached during saponification of a methyl or ethyl ester with lithium hydroxide in aqueous tetrahydrofuran, whereas Fmoc would be partially removed under those basic conditions. This is not an absolute rule; the exact stability depends on water content, temperature, and reaction time. Fmoc-L-tryptophan is preferred in solid-phase peptide synthesis on Wang or Rink amide resins because the deprotection step with 20% piperidine is fast and can be monitored by UV absorbance of the dibenzofulvene adduct. Boc-L-tryptophan is preferred in Boc SPPS where repeated trifluoroacetic acid deprotections are used. Cbz-L-tryptophan is less convenient for stepwise solid-phase synthesis because removal requires hydrogenation equipment and is not easily automated across multiple cycles. The comparative properties are summarized in Table 2.
| Property | CBZ-L-tryptophan | Fmoc-L-tryptophan | Boc-L-tryptophan |
|---|---|---|---|
| Primary deprotection condition | H2/Pd-C or HBr/acetic acid | 20% piperidine in DMF | 25–50% trifluoroacetic acid in DCM |
| Deprotection mechanism | Hydrogenolysis or acidolysis | β-Elimination via dibenzofulvene | Acidolysis |
| Stability in 20% piperidine/DMF | Retained over 30 min at 25 °C | Removed within 5–15 min | Retained |
| Stability in brief TFA treatment | Generally retained; removed by HBr/acetic acid | Retained | Removed |
| Hydrogenation compatibility | Removed by H2/Pd | Generally retained under mild H2/Pd | Generally retained under mild H2/Pd |
| Typical synthesis platform | Solution-phase convergent synthesis | Fmoc SPPS | Boc SPPS |
On a production scale, the handling of Cbz-L-tryptophan differs from Fmoc and Boc derivatives in one key aspect: hydrogenolytic deprotection generates toluene and carbon dioxide, which must be vented and separated from the catalyst. The use of a pressure-rated reactor with a gas-entrainment impeller and a catalyst filtration train with 0.5–5 µm retention avoids product contamination by palladium particles. Catalyst poisoning by sulfur-containing impurities in tryptophan-derived substrates is a known cause of extended batch times; a sulfur guard bed or a higher catalyst loading of 10 wt% may be required. Published data for a single optimized hydrogenation of CBZ-L-tryptophan on a multi-kilogram scale is limited; route development should therefore include a design-of-experiments study over 1–5 bar hydrogen pressure, 25–40 °C, and 5–10 wt% Pd/C to identify the minimum catalyst loading that reaches endpoint within an acceptable cycle time.
CBZ-L-tryptophan is activated at the C-terminus, not through the protected amino group. The most common industrial activation strategies use dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, with 1-hydroxybenzotriazole or ethyl cyanohydroxyiminoacetate as auxiliary nucleophiles. In a jacketed reactor controlled at 0–10 °C, CBZ-L-tryptophan is dissolved in dimethylformamide, and the carbodiimide is added as a solid or as a concentrated solution over 30–60 min. The temperature must remain below 10 °C during activation because L-tryptophan-derived oxazolones racemize more readily than aliphatic amino acid derivatives; the indole ring does not prevent this pathway. A carbodiimide-to-substrate ratio of 1.00–1.05 equiv and an auxiliary nucleophile ratio of 0.95–1.10 equiv relative to carbodiimide are typical. After activation for 10–20 min, the amine component is added. When the amine is a hydrochloride salt, an equivalent of a tertiary base such as N-methylmorpholine or diisopropylethylamine is added to liberate the free amine but should not exceed 1.1 equiv to avoid base-catalyzed racemization. The reaction is quenched with 5% aqueous citric acid or 0.5 N hydrochloric acid; the organic phase is washed with sodium bicarbonate and brine. Chiral HPLC of the final protected peptide after workup gives the enantiomeric excess; values below 99.0% usually trace to excess base, high activation temperature, or extended reaction time.
Cbz is not suitable if the peptide or intermediate contains a benzyl ester, a nitro group, an aryl halide, or another reducible moiety that cannot survive catalytic hydrogenation. In these cases, the synthetic route should replace Cbz with a protecting group that can be removed without reducing conditions—such as Fmoc when base lability is acceptable or Alloc when selective palladium-catalyzed allyl transfer is feasible—or the sequence should be redesigned so that the tryptophan residue is introduced after the reducible functionality is removed. CBZ-L-tryptophan is also not recommended for solid-phase synthesis on acid-labile resins if final cleavage is performed with hydrogen fluoride or triflic acid, because the Cbz group may undergo partial acidolysis under those harsh conditions. The indole side chain of tryptophan can undergo oxidation during prolonged hydrogenation or exposure to strong acid; common side products include oxindole and sulfenyl chloride adducts when using HBr/acetic acid that contains trace chlorine. For this reason, the final peptide should be monitored by LC-MS for +16 amu oxidation peaks and the indole UV signature at 280 nm should be retained.
