| HS Code | 794559 |
| Cas Number | 1218-34-4 |
| Ec Number | 214-937-9 |
| Molecular Formula | C13H14N2O3 |
| Molecular Weight | 246.26 g/mol |
| Iupac Name | (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid |
| Smiles | CC(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)O |
| Appearance | White to off-white crystalline powder |
| Melting Point | 187-190 °C |
| Solubility | Soluble in ethanol and DMSO; sparingly soluble in water |
| Storage Conditions | Store at 2-8 °C, protected from moisture and light |
| Purity | ≥98% (HPLC) |
| Optical Rotation | [α]D20 +27.0° (c=1, water) |
As an accredited N-acetyl-L-tryptophan factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Packaged as 25 g of N-acetyl-L-tryptophan in a sealed glass vial with tamper-evident cap and desiccant. |
| Container Loading (20′ FCL) | 20' FCL loading: N-acetyl-L-tryptophan packed in drums on pallets, approximately 20 metric tons per container, secured and moisture-protected. |
| Shipping | N-acetyl-L-tryptophan should be shipped in sealed, leak-proof containers at ambient temperature, protected from light, moisture, and extreme heat. It is not typically classified as dangerous goods, but packaging must be sturdy and compliant with transport regulations. Upon receipt, store in original, tightly closed container in a cool, dry place. |
| Storage | Store N-acetyl-L-tryptophan in a tightly sealed container in a cool, dry, well-ventilated area, protected from light and moisture. Avoid prolonged exposure to heat. For long-term storage, refrigeration may be advisable. Keep away from incompatible materials such as strong oxidizing agents and ensure the container remains tightly closed when not in use. |
| Shelf Life | Store in a cool, dry place away from light. Shelf life is typically 2–3 years when unopened. |
In amino acid API manufacturing, N-acetyl-L-tryptophan is processed as an acetyl-protected precursor that releases L-tryptophan upon enantioselective hydrolysis by L-aminoacylase (EC 3.5.1.14). The substrate is charged into a jacketed glass-lined reactor equipped with a retreat-curve impeller and a temperature control loop maintaining 37–45 °C; the pH is held at 7.0–7.8 by metered addition of 2 M NaOH because the reaction liberates one equivalent of acetic acid per mole of L-tryptophan formed. Typical batch records specify a substrate concentration of 50–150 g/L and an enzyme-to-substrate loading of 0.5–2.0% w/w; under these conditions conversion exceeds 95% within 8–16 h, but published data for any specific commercial acylase preparation should be confirmed against its enzyme activity certificate because immobilization supports alter apparent Km and diffusion constraints. The resulting hydrolysate is clarified by tangential-flow filtration across a 10 kDa polyethersulfone membrane to remove biocatalyst particulates, then concentrated under vacuum at ≤60 °C. L-tryptophan is precipitated by adjusting to pH 5.5–5.8, filtered, and recrystallized from aqueous ethanol; the isolated crystals are dried in a vacuum tray dryer at 50–60 °C to final moisture below 0.5%. Compliance testing follows the Ph. Eur. monograph for L-tryptophan and the corresponding USP monograph, with particular attention to residual acetic acid measured by gas chromatography and to related substances by liquid chromatography; N-acetyl-L-tryptophan itself is monitored as a residual substrate marker if conversion is incomplete.
Coupling of N-acetyl-L-tryptophan to resin-bound peptides in automated solid-phase synthesis supports the introduction of an acetylated N-terminal tryptophan residue without use of a separate acetic anhydride capping step. The indole side chain remains unprotected under these conditions; therefore, coupling is performed in amber glassware and the reaction vessel is blanketed with nitrogen to limit indole oxidation and chlorination during activation. A typical cycle on a 0.1 mmol scale peptide synthesizer uses 3 equivalents of N-acetyl-L-tryptophan, 3 equivalents of a 0.4 M uronium activator such as HCTU in dimethylformamide, and 6 equivalents of diisopropylethylamine, with single coupling for 60 min at 25–30 °C. The residual free amine is assessed by ninhydrin or chloranil test; if staining persists, a second coupling is executed. Cleavage from the resin with trifluoroacetic acid/ethanedithiol/water at 90:5:5 v/v/v for 2–3 h liberates the acetylated peptide bearing an N-terminal tryptophan residue; ethanedithiol is essential because the unprotected indole ring is susceptible to acid-catalyzed dimerization and oxidation during cleavage. The crude product is precipitated in cold diethyl ether, centrifuged, and purified by reversed-phase preparative HPLC on a C18 column with acetonitrile/water gradients containing 0.1% trifluoroacetic acid. Final purity is measured by HPLC according to the general principles of USP <621> and Ph. Eur. 2.2.29. For peptides containing multiple tryptophan residues, the use of N-acetyl-L-tryptophan as a discrete coupling reagent limits introduction of the final indole to one defined position, but published data for this specific configuration in process-scale peptide synthesizers is limited; laboratory-scale coupling efficiencies are typically above 95% by HPLC area normalization when the resin is adequately swollen and free base is removed by two dimethylformamide washes before activation.
Human albumin solutions require pasteurisation at 60.0 ± 0.5 °C for 10–11 h as a viral inactivation step; without stabilizers, this heat load causes irreversible aggregation and loss of monomer content. The pharmacopoeial strategy combines sodium caprylate and N-acetyl-DL-tryptophan; the monograph Ph. Eur. 0255 permits these stabilizers within defined limits, and the finished product is controlled for aggregate content by size-exclusion chromatography against monomer purity criteria. N-acetyl-L-tryptophan, the L enantiomer, binds to the hydrophobic binding cleft at Sudlow site II of human serum albumin through its indole ring, while the carboxylate group forms electrostatic interactions with basic residues; this ligand binding restricts heat-induced conformational flexibility and reduces exposure of hydrophobic surfaces that would otherwise associate. In commercial albumin manufacturing, the stabilizer solution is added after cold ethanol fractionation and before pasteurisation, with sodium caprylate concentrations commonly near 0.08 mmol/g protein and N-acetyl-DL-tryptophan near 0.08 mmol/g protein; the exact ratio is formulation-specific and must be confirmed against the marketing authorization dossier. The addition is performed in a stainless steel mixing vessel under controlled pH 6.4–7.4, and the bulk is then sterilized by filtration through a 0.2 µm membrane prior to aseptic filling after pasteurisation. Process challenges include photodegradation of the tryptophan stabilizer during storage if not protected from light, and the possibility of stabilizer oxidation in the presence of residual oxygen in the headspace; therefore, manufacturers use nitrogen-purged filling lines and amber primary packaging. For isolated N-acetyl-L-tryptophan as a sole stabilizer, published industrial stability data is limited because the racemic DL form remains the standard pharmacopoeial species; investigational formulations using the pure L form require chiral purity verification by HPLC with a chiral stationary phase.
When L-tryptophan API batches are released for use in parenteral nutrition or as pharmaceutical starting material, residual N-acetyl-L-tryptophan is monitored as an acetylated related substance because incomplete deacetylation during upstream synthesis or enzymatic resolution leaves trace quantities in the final crystals. A typical impurity-directed HPLC method uses a 250 × 4.6 mm octadecylsilane column with 5 µm particles, maintained at 25 °C, and a mobile phase prepared from phosphate buffer at pH 3.0 and acetonitrile in a gradient from 5% to 60% acetonitrile over 40 min; detection is set at 280 nm because both L-tryptophan and the N-acetyl derivative retain the indole chromophore. System suitability requires resolution between L-tryptophan and N-acetyl-L-tryptophan of not less than 2.0, and the reference solution is prepared at 0.1% relative to the nominal sample concentration to approximate the expected reporting threshold. Quantification is performed by area normalization with a correction factor determined from linearity solutions spanning 0.05–0.5% of the analyte. The identity of the N-acetyl-L-tryptophan peak is confirmed by comparing retention time and UV spectral purity against a certified reference standard; mass confirmation by liquid chromatography–mass spectrometry using negative-ion electrospray ionization detects the deprotonated molecular ion at m/z 245.1. This impurity tracking application is established in amino acid quality control laboratories, although published data for specific L-tryptophan manufacturing routes is limited because impurity profiles vary with the fermentation or chemoenzymatic pathway employed.
Because the indole side chain of tryptophan dominates the intrinsic fluorescence of albumin and many therapeutic proteins, N-acetyl-L-tryptophan is employed as a low-molecular-weight proxy to calibrate quenching responses and to isolate binding contributions at Sudlow site II. In a typical steady-state fluorescence titration, a 2.0 mL quartz cuvette containing human serum albumin at 2.0 µM in phosphate-buffered saline at pH 7.40 is titrated with 1.0–5.0 µL increments of a 5.0 mM N-acetyl-L-tryptophan stock solution; excitation is set at 295 nm to avoid tyrosine excitation, and emission is collected from 310 nm to 420 nm. The resulting Stern-Volmer plot is analysed using F = F₀/(1 + Ksv[Q]), where the quenching constant for site II occupancy is compared against the value obtained with the racemic stabilizer; differences in Ksv between the L and D enantiomers are small for hydrophobic site binding but become detectable when chiral additives occupy the same cleft. This application supports formulation screening for albumin-stabilized biotherapeutics and is not a release test; therefore, the data are interpreted as relative binding order rather than as a regulatory specification. For quantitative publication, the inner-filter effect at 295 nm excitation must be corrected because N-acetyl-L-tryptophan absorbs weakly at the excitation wavelength at the higher titrant concentrations; correction is performed using absorbance measured on a UV spectrophotometer according to Ph. Eur. 2.2.25 and the equation F_corr = F_obs × antilog[(A_ex + A_em)/2]. Published data for inter-laboratory reproducibility is limited, but the method is widely reported in peer-reviewed protein-ligand interaction studies.
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N-Acetyl-L-tryptophan is supplied as a white to off-white crystalline powder identified by the Chemical Abstracts Service registry number 1218-34-4, molecular formula C13H14N2O3, and molar mass 246.26 g/mol. The product is not associated with a single proprietary machine model; industrial purchase specifications refer instead to the CAS registry number, the pharmacopoeial test alignment, and the residual solvent profile. In chemical terms, the acetyl substituent is attached to the α-amino nitrogen of L-tryptophan, leaving the indole side chain intact. The blocked primary amine removes the principal site for Schiff base formation, Maillard browning, and diketopiperazine formation, while the indole chromophore retains absorbance near 280 nm and native fluorescence. The substance is therefore used as a heat-stabilising excipient in pasteurised human albumin solutions, as an N-terminal protected building block in peptide synthesis, and as a chiral reference for tryptophan degradation studies. Because acetylation does not protect the indole ring from oxidative ring opening, the material is not a universal substitute for L-tryptophan when photostability is the limiting formulation variable.
The primary structural difference is the absence of a free α-amino group. In L-tryptophan (CAS 73-22-3), the primary amine participates in condensation with reducing sugars and aldehydes. In N-acetyl-L-tryptophan (CAS 1218-34-4), the acetyl amide is less nucleophilic and does not enter Maillard chemistry under typical parenteral formulation conditions. The indole side chain remains present in both molecules; oxidative degradation products such as N-formylkynurenine and kynurenine are therefore still observed under forced light and oxidant exposure. The racemic sodium salt, N-acetyl-DL-tryptophan sodium, is the water-soluble stabiliser form commonly used in plasma-derived albumin solutions; the L-isomer free acid can be converted to the same stabiliser species by neutralisation with sodium hydroxide before addition to the albumin bulk. Chiral identity affects enantiomeric purity testing and the use of the material in peptide assembly, where the L-configuration must be preserved.
| Parameter | L-tryptophan | N-acetyl-L-tryptophan | N-acetyl-DL-tryptophan sodium |
|---|---|---|---|
| CAS registry number | 73-22-3 | 1218-34-4 | Supplied as racemic sodium salt; single-isomer CAS not used for the stabiliser mixture |
| Molecular formula | C11H12N2O2 | C13H14N2O3 | C13H13N2NaO3 |
| Molar mass | 204.23 g/mol | 246.26 g/mol | 268.25 g/mol |
| α-amino group | Free primary amine | Acetylated | Acetylated |
| Primary chemical incompatibility | Maillard condensation with reducing sugars | Strong oxidisers; indole ring remains sensitive to singlet oxygen | Strong oxidisers; salt form has higher aqueous solubility |
| Typical industrial use | Parenteral nutrition amino acid source | Heat-stabiliser, peptide intermediate, chiral reference | Albumin stabiliser after neutralisation to pH 7.0 |
Commercial release documentation usually reports assay by reversed-phase high-performance liquid chromatography, optical rotation, water content, and residue on ignition. Bulk density and tapped density are not compendial identifiers for this substance, but they are relevant when the powder is blended with other dry excipients for solid-dosage or diagnostic intermediates. The powder should be handled under nitrogen or dry inert gas when the ambient relative humidity exceeds 60%, because residual moisture uptake can bias the loss-on-drying result and reduce the accuracy of gravimetric dispensing in sealed transfer isolators. Polyethylene double-lined fibre drums with desiccant pouches are standard for kilogram-scale lots; packaging configuration is supplier-specific and should be reviewed for oxygen barrier performance.
The indole fluorescence emission maximum of N-acetyl-L-tryptophan in dilute aqueous buffer is near 350 nm when excitation is set at 280 nm; the emission signal is used in albumin stabiliser assays and ligand-binding fluorescence quenching experiments. Acetylation of the α-amino group has a smaller effect on emission wavelength than does solvent polarity, so the derivative remains useful as a probe for hydrophobic binding pockets. Differential scanning calorimetry of the fully dried free acid typically exhibits a melting endotherm near 188-190°C; L-tryptophan, by contrast, degrades near 290°C and does not provide the same sharp thermal signature.
In plasma-derived human albumin production, the isolated albumin fraction is formulated with sodium caprylate and N-acetyltryptophan sodium before sterile filtration and aseptic filling into final glass containers. The stabiliser concentration is typically 0.004 M for each component. The filled containers are heat-treated at 60 ± 0.5°C for 10 hours; this terminal pasteurisation step reduces viral risk and is described in human albumin monographs and in 21 CFR 640.80. N-Acetyl-L-tryptophan does not act as a simple antioxidant in this system. It binds reversibly to hydrophobic regions on albumin, limits heat-induced aggregation, and reduces the formation of insoluble polymers that would otherwise appear after prolonged heating. The stabiliser is not removed after pasteurisation; it remains in the finished product at the labelled concentration. To meet the 10-hour hold, chamber load validation includes cold-spot mapping with wired thermocouples and the hold timer starts only after the slowest-heating container reaches 60°C. In water-bath pasteurisation, glass bottles are submerged in HEPA-filtered water to reduce air thermal resistance; in steam-air pasteurisation, the chamber is maintained under positive pressure to keep closures sealed. Fill volume is limited so that the internal solution reaches the viral-inactivation temperature without excessive overshoot. Batch-to-batch variance in stabiliser binding is less affected by the optical rotation of the acetylated tryptophan than by the molar ratio of caprylate to albumin and by the residual lipid content of the albumin fraction. Published data for N-acetyl-L-tryptophan in recombinant albumin pasteurisation at this specific concentration is limited, but the same stabiliser system is widely used in plasma-derived albumin and is referenced in the European Pharmacopoeia monograph 0255 for human albumin solution.
During pasteurisation, the acetylated tryptophan may undergo partial oxidation if the final container headspace contains oxygen. Manufacturers therefore control dissolved oxygen in the albumin bulk and purge headspace with inert gas before sealing. High-performance liquid chromatography with ultraviolet detection at 280 nm resolves the parent stabiliser from more polar oxidation products, which appear under accelerated stability conditions at relative retention times characteristic of kynurenine-type ring-opening. The detected oxidation peaks are not necessarily process impurities; they can also be generated during sample preparation if the analyst exposes unstabilised aqueous samples to ambient laboratory light. Sample vials are prepared in amber glass and injected within a defined sequence interval to limit photochemical artefacts. Method transfer between laboratories often shows retention-time variability because the analyte is sensitive to mobile-phase pH and column silanol activity. A reversed-phase method using an end-capped C18 column with a phosphate-acetonitrile gradient at low pH produces acceptable peak symmetry; detection at 280 nm avoids the high baseline produced at 210-220 nm by residual acetic acid and buffer components. System suitability criteria typically require tailing factor between 0.8 and 1.5, plate count above 2000, and injection precision below 2.0% relative standard deviation for five replicate injections. These values are method-specific and are not universal compendial limits.
Replacement of L-tryptophan with N-acetyl-L-tryptophan in forced-degradation studies does not create a light-stable molecule; it changes the balance between amine-mediated degradation and indole oxidation. Under ICH Q1B photostability conditions, aqueous L-tryptophan can form brown condensation products when reducing sugars or aldehydes are present, whereas N-acetyl-L-tryptophan produces fewer of these amine-derived chromophores. The indole ring still undergoes oxidative ring opening to N-formylkynurenine and kynurenine, with relative amounts dependent on dissolved oxygen, pH, and light intensity. The absorbance of the indole chromophore near 280 nm means that ultraviolet-B and ultraviolet-A exposure can promote electron transfer from the aromatic system; the resulting degradation profile shifts to earlier eluting polar species under reversed-phase HPLC conditions. Users who require complete protection from photodegradation should use light-protected packaging and oxygen-free diluents, because acetylation alone does not eliminate photochemical vulnerability. Published quantitative quantum-yield data for N-acetyl-L-tryptophan in parenteral admixtures is limited; degradation rates should be measured under the specific container, light source, and dissolved oxygen level of the intended process.
At process scale, acylation of L-tryptophan with acetic anhydride is exothermic. The reaction is typically conducted in aqueous alkaline solution at 0-5°C with controlled addition of the acylating agent to minimise racemisation and thermal decomposition of the indole ring. After neutralisation and crystallisation, the product is washed to remove acetate and dried under vacuum. The final recrystallisation solvent system is supplier-specific, but common release tests include gas chromatographic residual solvent analysis according to USP <467> or Ph. Eur. 5.4. Ethanol and water are preferred crystallisation solvents when the material is intended for parenteral excipient use because they avoid the more toxic Class 2 solvents such as methanol or dichloromethane. If methanol is used earlier in the synthesis, the certificate of analysis should demonstrate compliance with the ICH Q3C limit of 3000 ppm for methanol or a tighter in-house limit. Production-scale filterability of the final crystallisation slurry is influenced by crystal habit; slow addition of the acid precipitant and controlled stirring reduce the formation of fine needles that can blind filter cloths and extend batch cycle time.
For N-acetyl-L-tryptophan, the pharmacopoeial control strategy is typically assembled from general chapters rather than from a dedicated substance monograph. The assay and related-substances determination commonly follows USP <621> liquid chromatography, with method parameters and impurity reference standards defined by the supplier. Optical rotation is measured according to USP <781> or Ph. Eur. 2.2.7; the positive rotation confirms the L-configuration, but the result must be corrected for solvent and moisture. The table below lists representative quality attributes and test method alignments used by industrial suppliers; these are not official monograph limits.
| Quality attribute | Method alignment | Representative release acceptance criterion |
|---|---|---|
| Appearance | Visual inspection | White to off-white crystalline powder |
| HPLC assay | USP <621> / Ph. Eur. 2.2.29 | ≥98.0% area normalised |
| Specific rotation | USP <781> / Ph. Eur. 2.2.7 | +24° to +29° at 20°C |
| Loss on drying | USP <731> / Ph. Eur. 2.2.32 | ≤0.5% |
| Residual solvents | USP <467> / Ph. Eur. 5.4 | ICH Q3C limits for the production solvents used |
| Elemental impurities | USP <232>/<233> | ICH Q3D limits for parenteral use |
Compared with 5-hydroxy-L-tryptophan, N-acetyl-L-tryptophan is not a serotonin precursor because the indole ring is not hydroxylated at the 5-position and the acetylated amino group is not available for decarboxylation. This distinction is relevant when tryptophan derivatives are used in cell-culture media or in enzyme-substrate studies; the acetylated derivative should not be assumed to support serotonin biosynthesis. Compared with N-acetyl-L-tyrosine, the tryptophan derivative is more lipophilic because of the indole ring, and its aqueous solubility as the free acid is pH-dependent; neutralisation to the sodium salt increases solubility and is the preferred form for parenteral stabiliser addition.
In formulation development, N-acetyl-L-tryptophan should not be considered interchangeable with free L-tryptophan on a molar basis without verifying the effect on osmolality and nitrogen contribution. The acetylated derivative contributes an additional carbonyl and methyl group that alter polar surface area and log P relative to L-tryptophan; the blocked amino group also means it cannot serve as a substrate for peptide condensation without a deprotection step. When the material is used as a stabiliser in albumin-containing solutions, the final product monograph controls the total stabiliser content by high-performance liquid chromatography or ultraviolet detection. The most relevant incompatibility is with strong oxidising agents and with reactive aldehyde impurities in excipients; the indole ring remains reactive toward electrophilic substitution and oxidative cleavage. For dry applications, the powder is best stored at controlled room temperature below 25°C, protected from light and moisture, and retested at the interval assigned by the supplier. Suitability must be established by stability studies in the intended formulation.
