| HS Code | 992704 |
| Chemical Name | N-Acetyl-L-tyrosine Ethyl Ester Monohydrate |
| Cas Number | 36546-50-6 |
| Molecular Formula | C13H17NO4·H2O |
| Molecular Weight | 269.29 g/mol |
| Appearance | White crystalline powder |
| Melting Point | 121-125 °C |
| Purity | ≥98% (TLC) |
| Solubility | Soluble in ethanol, methanol, and DMSO; sparingly soluble in water |
| Specific Rotation | [α]20/D +20.0° (c = 2% in ethanol) |
| Storage Temperature | 2-8 °C |
| Water Content | 6.69% (w/w) theoretical |
| Ec Number | 244-239-2 |
| Mdl Number | MFCD00002234 |
As an accredited N-Acetyl-L-tyrosine Ethyl Ester Monohydrate 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-tyrosine Ethyl Ester Monohydrate in a sealed glass bottle with desiccant. |
| Container Loading (20′ FCL) | 20' FCL container loading: N-Acetyl-L-tyrosine Ethyl Ester Monohydrate packed in drums, secured, ventilated, dry, temperature-controlled, chemical-safe conditions. |
| Shipping | N-Acetyl-L-tyrosine Ethyl Ester Monohydrate ships in sealed, moisture-proof containers to preserve stability. Store at 2–8°C, away from light and humidity. Handle with standard lab precautions—avoid dust inhalation and skin contact. Transport under ambient conditions if protected from heat and moisture; ensure compliance with local chemical shipping regulations. |
| Storage | Store N-Acetyl-L-tyrosine ethyl ester monohydrate at -20°C in a tightly sealed container, protected from light and moisture. Keep desiccated, ideally under inert gas (nitrogen/argon) for long-term stability. Avoid repeated freeze-thaw cycles; warm to ambient temperature before opening to prevent condensation. Handle with standard laboratory precautions. |
| Shelf Life | Store in a cool, dry place, protected from light and moisture. Shelf life is typically 2–3 years when sealed properly. |
In pancreatic enzyme release testing, chymotrypsin activity is quantified through esterolytic hydrolysis of N-acetyl-L-tyrosine ethyl ester monohydrate. The substrate is dissolved in 0.08 M Tris-HCl buffer, pH 7.0, containing 0.05 M calcium chloride at 25°C. Dissolution completes in 2–5 minutes when the monohydrate is pre-sieved through a 180 µm screen. The enzymatic hydrolysis releases N-acetyl-L-tyrosine and ethanol. The reaction is followed by measuring the increase in absorbance at 237 nm. Initial-rate data are recorded every 30 seconds over a 180-second window. One USP Chymotrypsin Unit is defined as the amount of enzyme that hydrolyses 1 µmole of ATEE per minute at 25°C and pH 7.0 under the official monograph conditions. The assay remains linear only when substrate conversion is kept below 10%. Solutions are used within 60 minutes of preparation because slow autohydrolysis and alkaline pH drift produce background absorbance drift. Batch acceptance requires linear regression of absorbance against time with a correlation coefficient not lower than 0.995. Published inter-laboratory repeatability data for this specific monohydrate grade is limited. The dissolution time and initial absorbance are verified before adding enzyme to control batch-to-batch variance.
| Parameter | Specification | Method |
|---|---|---|
| Buffer | 0.08 M Tris-HCl, pH 7.0, 0.05 M CaCl₂ | pH meter calibrated at 25°C |
| Substrate level | 0.5–1.0 mM | UV absorbance at 237 nm |
| Conversion limit | <10% | initial-rate linearity |
| Temperature | 25°C ±0.1°C | thermostatted cell holder |
| Correlation coefficient | ≥0.995 | linear least-squares |
The N-acetyl group is not removable under standard Fmoc/t-Bu solid-phase peptide synthesis conditions. The cap resists 20% piperidine in dimethylformamide and trifluoroacetic acid cleavage cocktails. If the material is introduced as a protected tyrosine monomer, the N-acetyl group terminates the growing peptide chain at the point of incorporation. The compound is therefore not a viable SPPS building block for internal tyrosine residues. Its peptide chemistry use is limited to solution-phase condensations at the carboxyl terminus. The ethyl ester is first saponified with 1.0 M sodium hydroxide in aqueous acetone at 0–5°C for 45–60 minutes. The resulting N-acetyl-L-tyrosine is precipitated by acidification to pH 2.0 with 2 M hydrochloric acid. The dried acid is activated with dicyclohexylcarbodiimide and 1-hydroxybenzotriazole in dimethylformamide at -10°C. The resulting benzotriazolyl ester is coupled to the amino terminus of a peptide fragment. Residual water from the monohydrate must be removed before activation. Pre-drying over phosphorus pentoxide in vacuo at 35°C for 12 hours is required when tetrahydrofuran is the coupling solvent. Residual moisture above 0.1% reduces active ester concentration and may lower coupling yield below 80% for fragments longer than 20 residues. Published data for longer-fragment condensations with this specific derivative is limited.
Preparative hydrolytic conversion to N-acetyl-L-tyrosine represents the main non-analytical downstream use. The monohydrate is suspended in deionized water at 5–8% w/v. The suspension is adjusted to pH 11.5 with sodium hydroxide and held at 22–28°C for 2 hours under low-shear overhead stirring. The pH must remain above 11.0 during the hold; below pH 10.5 saponification of the ethyl ester falls below 10% conversion. The reaction mixture is then acidified to pH 2.5 with hydrochloric acid and aged at 4°C for 4 hours to precipitate N-acetyl-L-tyrosine. The crude solid is filtered, washed with cold acetone, and vacuum-dried at 40°C to residual moisture below 0.5%. N-acetyl-L-tyrosine recovered by this route is evaluated as a high-solubility tyrosine source for parenteral nutrient admixtures. Final injectable material must satisfy ICH Q3D elemental impurity limits, bacterial endotoxin below 0.5 EU/mg, and particulate matter requirements under USP <788> or an equivalent pharmacopoeial chapter. The monohydrate starting material is not used directly in the admixture because the ethyl ester is not an approved injectable amino acid derivative. The ester hydrolysis step is documented by HPLC assay with residual starting material below 0.1% unless the downstream purchaser defines a different acceptance criterion.
| Route | pH/temperature | Time | Residual starting material | Use case |
|---|---|---|---|---|
| Alkaline saponification | pH 11.5, 22–28°C | 2 h | <0.1% by HPLC after acidification | Preparative recovery |
| Enzymatic chymotrypsin hydrolysis | pH 7.0, 25°C | assay-limited; preparative run requires enzyme loading | variable; published preparative data limited | Kinetic studies, chiral confirmation |
| Acid-catalyzed ester cleavage | not recommended | not specified | risk of acetyl loss | analytical comparison only |
Inhibitor screening uses initial-velocity conditions similar to compendial assay geometry but with controlled ionic strength. ATEE is dissolved in 50 mM Tris-HCl, pH 7.0, containing 0.15 M sodium chloride. The fixed substrate concentration is 0.8 mM unless a full Michaelis-Menten curve is being generated. Test compounds are pre-incubated with chymotrypsin for 10 minutes at 25°C before the substrate is added. Hydrolysis is monitored at 237 nm in a 96-well UV-transparent plate with pathlength correction. The maximum DMSO concentration in the final well must not exceed 1% v/v because higher DMSO levels alter chymotrypsin conformation. Kinetic readings are collected every 15 seconds for 5 minutes. Inhibition constants are calculated from linear regression of the initial-rate region. Published Km values for bovine pancreatic chymotrypsin with ATEE are generally reported in the range 0.7–1.4 mM; the exact value shifts with calcium and chloride concentration. The monohydrate stock solution is prepared fresh daily because slow autohydrolysis produces N-acetyl-L-tyrosine and ethanol, which can create background drift after storage beyond 24 hours.
The L-tyrosine core remains intact during both alkaline and enzymatic hydrolysis. Racemisation is controlled when saponification pH is maintained below 12.0 and temperature below 30°C. The recovered N-acetyl-L-tyrosine is used as a chiral pool building block for further synthesis of substituted tyrosine analogues. Enzymatic hydrolysis by chymotrypsin is stereospecific and does not cleave the D-ester. This selectivity can be used to detect chiral contamination when a D-enriched sample is spiked into the L-ester. The detection limit for D-ester contamination is not fixed in pharmacopoeial monographs; method validation is performed by the receiving laboratory using a D-ester reference standard. For preparative chiral recovery, alkaline hydrolysis is preferred because it avoids residual protein contamination. The isolated N-acetyl-L-tyrosine is recrystallised from hot water and dried below 0.5% moisture. Optical rotation is confirmed at 25°C in water against a certified reference. Published data for multi-kilogram isolated yield from this specific monohydrate is limited.
N-Acetyl-L-tyrosine ethyl ester monohydrate can be used as a reference substrate to distinguish chymotrypsin activity from non-specific esterase activity in finished enzyme blends. The assay uses the same 237 nm detection wavelength but omits calcium chloride from the buffer. Calcium ions activate chymotrypsin but have less effect on many non-specific esterases. A parallel assay with and without 0.05 M calcium chloride produces an activity ratio. A ratio above 1.5 indicates a calcium-sensitive chymotrypsin component; a ratio near 1.0 indicates dominant non-specific esterase activity. The procedure is not a compendial method and must be validated by the receiving laboratory. Critical interferences include turbidity from undissolved monohydrate particles below 10 µm in the stock solution. Filtration through a 0.45 µm polyethersulfone membrane is required before use. The filtered solution remains stable for 8 hours at 4°C. Long-term storage of the dry monohydrate requires sealed containers below 25°C and below 60% RH because the monohydrate is hygroscopic and may deliquesce.
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N-Acetyl-L-tyrosine ethyl ester monohydrate, CAS 36546-50-6, is distributed as a white to faintly cream crystalline powder with the molecular formula C13H17NO4·H2O and a molar mass of 269.29 g/mol. The structure contains an acetylated α-amino group, an ethyl ester at the carbonyl terminus, and a free 4-hydroxyl on the phenyl ring. The stoichiometric monohydrate carries one water molecule per molecule of ester, corresponding to a theoretical water content of 6.69% w/w. The substance is supplied primarily as a blocked amino acid derivative for peptide synthesis, as a substrate in α-chymotrypsin activity assays, and as a synthetic intermediate in pharmaceutical research. Ordering is by name and CAS registry number rather than by a universal model code, because catalogs from major reagent suppliers apply their own SKU designations while the chemical identifier remains CAS 36546-50-6 with grade qualifiers such as research grade or reagent grade. A compendial monograph for the specific monohydrate may be absent; the certificate of analysis therefore becomes the controlling specification for lot acceptance.
Because the hydrate stoichiometry links water content to molecular weight, the certificate of analysis should state whether assay values are expressed on an anhydrous basis or as-is basis. Analytical release data for bulk lots typically derive from pharmacopeial general chapters even when no monograph is published for the exact ester monohydrate. The release matrix below lists the minimum test set used by several reagent suppliers for material labeled at 98.0% or higher purity.
| Test Parameter | Method | Compendial Reference | Typical Release Criterion |
|---|---|---|---|
| Appearance | Visual comparison | Internal QA procedure | White to off-white powder; no visible foreign matter |
| Infrared identification | KBr pellet or ATR | USP ⟨197K⟩ | Spectrum concordant with reference standard |
| Chromatographic purity | HPLC-UV at 220 nm | USP ⟨621⟩ | ≥98.0 area% on anhydrous basis |
| Water content | Coulometric Karl Fischer | USP ⟨921⟩ | 5.8–7.2% w/w |
| Residue on ignition | Sulfated ash | USP ⟨281⟩ | ≤0.1% |
| Residual solvents | Headspace GC | USP ⟨467⟩ / ICH Q3C | Class 2 and Class 3 solvents within ICH limits |
| Optical rotation | Polarimetry | USP ⟨781⟩ | Report value against certificate of analysis |
The acceptance bracket of 5.8–7.2% water includes both lattice water and small amounts of surface moisture. Batches falling outside this range should be evaluated for hydrate loss or moisture sorption before use in stoichiometric reactions, because the molecular-weight correction from monohydrate to anhydrous changes from 0.933 at the theoretical hydrate to 0.950 at 5.0% water content and 0.920 at 8.0% water content. For a molar preparation, 269.29 g of the monohydrate is required to prepare 1.00 L of a 1.00 M solution, whereas the anhydrous ester would require only 251.28 g.
Replacement of the carboxylic acid with an ethyl ester eliminates the carboxylate pKa near 2.2 and reduces the number of ionizable groups from two to one under pharmaceutical formulation conditions. The remaining phenolic hydroxyl has a pKa near 10.1, so the molecule remains largely neutral at pH 6.0–8.0. Compared with N-acetyl-L-tyrosine, the ethyl ester displays higher retention on a 5 µm C18 column using a mobile phase of acetonitrile and 0.1% phosphoric acid, consistent with lower polarity. Quantitative comparative log P data across all supplier grades is limited. In non-aqueous coating or solvent-based systems, the ester form avoids charged carboxylate interactions with cationic lipids and reduces the probability of ion-pairing with quaternary ammonium excipients. Unlike L-tyrosine ethyl ester hydrochloride, the N-acetyl derivative has no free amine, so Schiff base formation with aldehyde-containing excipients is suppressed.
| Property | L-Tyrosine | N-Acetyl-L-Tyrosine | N-Acetyl-L-Tyrosine Ethyl Ester Monohydrate |
|---|---|---|---|
| Molar mass | 181.19 g/mol | 223.23 g/mol | 269.29 g/mol |
| Free α-amine | Yes | No | No |
| Free carboxylic acid | Yes | Yes | No; ethyl ester |
| Phenolic hydroxyl pKa, approximate | 10.1 | 10.1 | 10.1 |
| Typical C18 retention | Low | Moderate | Higher |
| Hydrolysis susceptibility in aqueous buffer | Not applicable | Amide hydrolysis is slow under acid/base | Ethyl ester hydrolyzes under strong acid/base or enzymatic turnover |
Preparation of an aqueous substrate solution requires attention to pH- and temperature-dependent hydrolysis. A typical enzyme-kinetic preparation uses a 1.0 mM solution in 50 mM Tris-HCl buffer at pH 7.8 containing 10 mM calcium chloride. The monohydrate is dissolved at 25–30 °C with gentle agitation until clear. Solubility in pure water is limited; therefore, the buffer component and weakly alkaline pH are used to aid dissolution. The solution should be used within 4 h when held on ice, because spontaneous ester hydrolysis increases at pH values above 8.0 and below 2.0. Published kinetic stability data for all possible matrices is limited; site-specific validation should include HPLC monitoring of N-acetyl-L-tyrosine formation at 220 nm over the intended use period.
When the monohydrate is stored in a freezer and repeatedly opened in an uncontrolled atmosphere, condensation can deposit on the powder surface and alter water content. At ambient relative humidity above 60%, the powder may sorb atmospheric moisture beyond the stoichiometric lattice water; Karl Fischer analysis will overreport water relative to the theoretical monohydrate value. A dry-nitrogen purge or desiccated glovebox is therefore required during long dispensing campaigns. The material should be stored at −20 °C in tight, light-resistant containers; a 2–8 °C secondary condition is acceptable for short-term handling, but the risk of ester hydrolysis increases if the container is not resealed with a desiccant packet. Avoid combining the ester with strong acid or alkaline carriers, acid chlorides, anhydride crosslinkers, or oxidizing agents. The free phenolic hydroxyl may react with acylating agents; therefore, acylation reactions should be designed to consume the phenol selectively or use a protected intermediate.
If anhydrous conditions are necessary, vacuum drying at 40–60 °C can remove surface water, but heating above the hydrate dissociation temperature risks partial dehydration and partial hydrolysis. Published moisture-sorption isotherms for this monohydrate are limited; therefore, weigh-labeled stability chambers with calibrated dew-point sensors should be used when establishing an internal drying protocol. A Karl Fischer coulometer with a diaphragm electrode is typically used for routine water content verification after drying. The developer must separately verify that drying does not reduce hydrolytic assay recovery or increase free N-acetyl-L-tyrosine content beyond the release limit.
In α-chymotrypsin activity determinations, the ethyl ester serves as an ester substrate whose cleavage releases N-acetyl-L-tyrosine and ethanol. The reaction is monitored by the increase in ultraviolet absorbance at 237 nm, where the phenolic chromophore of the hydrolysis product absorbs. One international unit is frequently defined as the amount of enzyme that hydrolyzes 1.0 µmol of N-acetyl-L-tyrosine ethyl ester per minute at 25 °C and pH 7.8; however, unit definitions differ among suppliers, so the exact buffer molarity, calcium concentration, and path length must be recorded. Initial rate data should be collected during the first 2–5 minutes before ethanol accumulation, pH drift, or substrate depletion introduces nonlinearity. Non-enzymatic hydrolysis can be estimated from a blank run without enzyme; if the blank rate exceeds 5% of the enzyme-catalyzed rate, the buffer pH or solution age should be re-examined.
Compared with N-acetyl-L-tyrosine, the ethyl ester is cleaved rapidly by α-chymotrypsin; the free acid is not a substrate for the esterase activity. Compared with the methyl ester, the ethyl ester shows slower spontaneous hydrolysis under equivalent buffer conditions due to steric hindrance at the ester carbonyl. Published comparative turnover numbers require enzyme-specific validation, because variations in calcium concentration, ionic strength, and enzyme source can shift the apparent Michaelis constant. The assay blank should include the same concentration of monohydrate, buffer, and organic co-solvent if used; dimethyl sulfoxide at 5% v/v may be required to prepare a concentrated stock before dilution into buffer, but its effect on initial rate must be evaluated against a co-solvent-free control.
