| HS Code | 963665 |
| Chemical Name | L-Alanine tert-butyl ester hydrochloride |
| Cas Number | 39825-33-7 |
| Molecular Formula | C7H15NO2·HCl |
| Molecular Weight | 181.66 g/mol |
| Appearance | White to off-white crystalline powder |
| Melting Point | 174-176 °C |
| Solubility | Soluble in water, methanol, ethanol, and dichloromethane |
| Optical Rotation | [α]20D = -8.0° to -10.0° (c=2, water) |
| Purity | >98% (Titration/HPLC) |
| Storage Conditions | Store under inert atmosphere, in a cool dry place, protected from moisture, at 2-8 °C |
As an accredited L-alanine tert-butyl Ester Hydrochloride factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 25 g in an amber glass bottle with a sealed cap, labeled clearly, stored under nitrogen to maintain purity. |
| Container Loading (20′ FCL) | 20′ FCL: L-alanine tert-butyl ester hydrochloride loaded in drums/pallets, secured, ventilated, and labeled per chemical transport safety regulations. |
| Shipping | L-alanine tert-butyl Ester Hydrochloride is shipped in sealed, moisture-resistant containers under ambient temperature. Avoid exposure to heat, light, or humidity. Handle with standard laboratory precautions, using protective gloves and eyewear. Ensure compliance with local regulations for transportation of fine chemicals. |
| Storage | Store L-alanine tert-butyl ester hydrochloride in a tightly sealed container under an inert atmosphere, protected from moisture and light. Recommended storage is refrigerated at 2–8°C in a cool, dry place. Avoid exposure to air and humidity, as the compound is hygroscopic. Bring to room temperature before opening to prevent condensation. |
| Shelf Life | Store at -20°C under inert gas, desiccated. Shelf life: 2 years from manufacture if unopened. |
For C-terminal chain extension in solution-phase fragment coupling, L-alanine tert-butyl ester hydrochloride is charged as the C-terminal building block after vacuum drying at 40 °C and 10 mbar for 12 h; residual water in the dried salt is reduced to ≤ 0.1 wt% by Karl Fischer titration in accordance with USP ⟨921⟩. The dried salt is dissolved or suspended in anhydrous DMF or dichloromethane at 0–5 °C before coupling. In a representative glycine-alanine fragment assembly, N-fluorenylmethoxycarbonyl-glycine is pre-activated with 1.05–1.10 mol equiv of HATU and 2.5–3.0 mol equiv of DIPEA relative to the acid at −5 °C; the hydrochloride is then added at 1.00–1.05 mol equiv relative to the N-protected acid, and the mixture is stirred for 6–18 h with gradual warming to 15–20 °C. The tertiary-butyl ester remains intact when the pH of any aqueous wash is maintained between 3.5 and 6.5, because prolonged contact with aqueous base above pH 8 promotes ester hydrolysis and formation of alanine free acid. Workup includes dilution with ethyl acetate, successive washes with 5 wt% citric acid, 5 wt% sodium bicarbonate, and 20 wt% sodium chloride, followed by drying over anhydrous sodium sulfate and solvent displacement into methyl tert-butyl ether. The isolated product is an N-protected dipeptide tert-butyl ester such as Fmoc-Gly-Ala-OtBu or Cbz-Gly-Ala-OtBu, which is subsequently used in fragment condensation to longer peptide APIs. Process control is performed by reversed-phase HPLC with UV detection at 210–220 nm; if crude purity falls below 90 area%, the material is purified by silica flash chromatography using an ethyl acetate/heptane gradient. Under ICH Q7, this step is managed as a registered intermediate operation with defined cleaning validation, batch record review, and change control for coupling reagent suppliers.
The hydrochloride salt is converted to the free amine prior to N-acylation by partitioning between dichloromethane and aqueous sodium bicarbonate at 0–5 °C. The bicarbonate charge is set at 1.05–1.10 molar equivalents relative to the hydrochloride, which controls the aqueous phase pH between 7.5 and 8.0; higher pH values accelerate ester hydrolysis at the monomer stage, while for dipeptide fragments the parallel risk is intramolecular diketopiperazine formation. The free amine is extracted into the organic phase, dried over anhydrous sodium sulfate, and concentrated at ≤ 30 °C under ≤ 250 mbar to minimize thermal de-esterification. The concentrated free amine is then treated with Fmoc-OSu at 1.03–1.08 molar equivalents and DIPEA at 1.05–1.15 equivalents in dichloromethane or THF at 0–5 °C for 4–8 h. The terminal product is Fmoc-L-alanine tert-butyl ester, a protected building block used in solid-phase peptide synthesis and fragment coupling. Residual solvent removal from the final product is controlled against ICH Q3C; dichloromethane must be below 600 ppm, DMF below 880 ppm, and methanol below 3000 ppm in the released intermediate. The following matrix summarizes the solvent limits applied to released batches where these solvents are used in the neutralization and Fmoc protection sequence.
| Solvent | Class | PDE | Concentration limit |
|---|---|---|---|
| Dichloromethane | 2 | 6.0 mg/day | 600 ppm |
| N,N-Dimethylformamide | 2 | 8.8 mg/day | 880 ppm |
| Methanol | 2 | 30.0 mg/day | 3000 ppm |
| Ethyl acetate | 3 | 50 mg/day | 5000 ppm |
In continuous-flow process development for solution-phase peptide coupling, L-alanine tert-butyl ester hydrochloride is fed as a pre-cooled suspension in 2-methyltetrahydrofuran/DMF mixtures, because full dissolution at high concentration generates back-pressure drift across the PFA coil. The feed composition is typically 0.8–1.2 M hydrochloride in 80:20 v/v 2-MeTHF/DMF, while the acid feed contains 1.05 equivalents of the N-protected amino acid and 1.10 equivalents of HATU relative to the hydrochloride. A third feed stream delivers DIPEA at 2.5–3.0 equivalents relative to the acid, and the three streams are mixed in a glass or PTFE T-mixer with an upstream static mixer. The reaction coil has an internal diameter of 0.75–1.00 mm and a residence time of 30–120 s at 20–35 °C; back-pressure is maintained at 5–7 bar to suppress degassing from carbodiimide-derived by-products. In-line UV/Vis or FTIR monitoring of the amide carbonyl region at 1630–1700 cm⁻¹ is used to confirm steady-state conversion, and the outlet stream is quenched into aqueous citric acid to stop residual active ester. The terminal product is an N-protected alanine-containing peptide fragment with the tert-butyl ester intact, ready for further elongation or C-terminal deprotection. Operation at temperatures above 40 °C is avoided because the tert-butyl ester undergoes acid-catalyzed elimination to isobutylene and alanine-related impurities; therefore, the jacket or thermostat control is set with a deviation alarm at ±2 °C. The control strategy for this continuous step follows ICH Q13 for continuous manufacturing, including defined residence time distribution characterization and diversion valves for out-of-specification material. Published data on this specific ester hydrochloride in continuous-flow configurations is less extensive than batch data, so scale-down qualification must include verification of feed solids settling and pressure-drop stability over 6–8 h campaigns.
Cbz-L-alanine tert-butyl ester, prepared from the hydrochloride by acylation with benzyl chloroformate, is deprotected by catalytic hydrogenolysis to provide the free amine or the corresponding hydrochloride for downstream fragment coupling. The substrate is dissolved in ethyl acetate:methanol 4:1 v/v at 0.10–0.20 M, and 5% Pd/C or 10% Pd/C is charged at 5–10 wt% relative to the Cbz intermediate. The hydrogenation is run in a stirred pressure vessel at 3–5 bar hydrogen pressure and 20–25 °C for 1–3 h; over-extended reaction times beyond 6 h increase the risk of N-methylation or diketopiperazine formation if free amine is present. The catalyst is removed by filtration through a 0.45 µm PTFE membrane or depth filter, and the filtrate is acidified with hydrogen chloride in ethyl acetate at 1.0–1.05 molar equivalents to precipitate L-alanine tert-butyl ester hydrochloride as a white solid. The terminal product is the regenerated tert-butyl ester hydrochloride, which can be used in subsequent couplings where the hydrochloride is the preferred input form. The residual palladium content of the isolated solid is controlled by inductively coupled plasma mass spectrometry to meet ICH Q3D, with a typical oral PDE of 100 µg/day applied to the intermediate if it enters an API chain. Methanol and ethyl acetate are removed to ≤ 3000 ppm and ≤ 5000 ppm respectively before the material is released. Hydrogenation equipment is maintained with inert gas purging, and the wet catalyst is not allowed to contact dry chlorinated solvent under vacuum, which can generate localized exotherms. This specific hydrogenolysis route is preferred when orthogonal C-terminal protection is required and the N-protecting group is removed without exposing the ester to acid.
In anhydrous acylation of L-alanine tert-butyl ester hydrochloride with acid chlorides or active succinimidyl esters, the water content of the reaction solvent is controlled below 0.05 wt% by Karl Fischer titration before charging. At water levels between 0.05 wt% and 0.10 wt%, the hydrolysis rate of the tert-butyl ester produces carboxylic acid impurities that accumulate during the final aqueous workup and reduce isolated yield by 15–30% in small-scale neutralizations; above 0.10 wt%, the acid chloride is consumed by hydrolysis and the free amine may be re-protonated, causing incomplete acylation. The acylation is carried out in a jacketed glass-lined or Hastelloy C22 reactor equipped with pressure-vacuum nitrogen purging and a bottom drain valve. The hydrochloride is slurried in anhydrous dichloromethane or toluene at −10 to 0 °C, and the base is charged at 2.0–2.2 molar equivalents relative to the acid chloride to neutralize both the hydrochloride and the acid by-product. The acid chloride is added at 1.00–1.05 molar equivalents relative to the hydrochloride, keeping the internal temperature below 5 °C during the exothermic neutralization. The terminal product is an N-acylated alanine tert-butyl ester such as N-acetyl-L-alanine tert-butyl ester or N-benzoyl-L-alanine tert-butyl ester, used as a chiral intermediate in further synthesis. After addition, the batch is warmed to 15–20 °C and washed with ice-cold 5 wt% sodium bicarbonate only after full acid chloride consumption is confirmed by in-stream IR at 1780–1800 cm⁻¹. The organic phase is dried and the product crystallized from methyl tert-butyl ether/heptane. Pre-drying of the hydrochloride at 40 °C under 10 mbar for 12 h is required if ambient storage relative humidity exceeds 60%. This process boundary is particularly important in multi-purpose facilities where the same dryer rotates between solvent-wet intermediates.
The final isolation of L-alanine tert-butyl ester hydrochloride from reaction mixtures or as a supplied starting material involves controlled crystallization from methyl tert-butyl ether/heptane blends and vacuum drying. The crude salt is dissolved at 35–40 °C in methyl tert-butyl ether containing 1.0–2.0 equivalents of hydrogen chloride relative to residual free amine, then cooled to −5 to 0 °C for 4–6 h. The crystalline solid is filtered, washed with pre-cooled 1:5 v/v methyl tert-butyl ether/heptane, and dried at 35 °C under ≤ 10 mbar for 8–16 h. The isolated hydrochloride is analyzed by acid-base titration with perchloric acid in accordance with USP ⟨541⟩; assay acceptance is set at 98.0–102.0% on the anhydrous basis. Loss on drying by USP ⟨731⟩ is controlled at ≤ 0.5%, and specific rotation is measured at 589 nm and 20 °C in methanol at c=1 to verify enantiomeric integrity. Residual solvents are reported against ICH Q3C, and elemental impurities are reported against ICH Q3D with validated inductively coupled plasma mass spectrometry methods. The terminal product is a white to off-white crystalline powder used directly as a C-terminal peptide building block or as a salt for generating the free amine in situ. Batches are stored in double polyethylene liners inside fiber drums, at 20–25 °C and below 40% RH, to reduce hygroscopic caking. If cake formation occurs, the batch is not milled without low-humidity nitrogen because mechanical shear can generate static charge and solvent retention. Published data on caking rates under high humidity is limited, but production facilities commonly observe agglomeration above 60% RH within 48 h in unsealed containers.
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L-Alanine tert-butyl ester hydrochloride (CAS 13404-22-3, molecular formula C7H16ClNO2, formula weight 181.66 g mol⁻¹) is the crystalline hydrochloride salt of the tert-butyl ester of L-alanine. It is supplied as a white to off-white powder or granular solid and is used as a C-terminal protected amino acid intermediate in solution-phase peptide synthesis and chiral amine manufacture. The hydrochloride form is preferred over the free ester for warehouse handling because it is non-volatile, less sensitive to atmospheric carbon dioxide, and can be weighed without cold-chain handling during short transfer operations. The free amine is released in situ with a tertiary base immediately before acylation. The tert-butyl ester is installed to provide acid-selective carboxyl protection; it is cleaved by trifluoroacetic acid in dichloromethane under conditions that leave N-terminal Fmoc or Boc groups largely intact, whereas methyl and ethyl esters generally require saponification or strong aqueous acid and may expose N-protecting groups to undesired hydrolysis. No unified industrial model number exists for the compound; commercial catalogue numbers are assigned by the supplier and should be linked to lot-specific certificates of analysis. Common pack sizes are 25 g, 100 g, 1 kg, and bulk quantities under nitrogen.
Representative acceptance criteria from commercial certificates of analysis are listed in Table 1. The assay is normally determined by reversed-phase HPLC with UV detection at 210 nm; the area-normalised purity acceptance is typically ≥98.0%. Water is determined by Karl Fischer titration according to USP 921 or Ph Eur 2.5.12; the limit is usually ≤0.5% because water accelerates ester hydrolysis and promotes caking. Enantiomeric purity is assessed by chiral HPLC rather than specific rotation alone; an acceptance of ≥99.0% enantiomeric excess is common. The chloride content should agree with the theoretical value of 19.52% by weight, with a typical acceptance window of 18.8–20.2%. Residual solvents are controlled by headspace gas chromatography according to USP 467 procedure A. Melting point is not used as a primary release criterion because the hydrochloride salt may exhibit broad decomposition endotherms that vary with heating rate. The material is hygroscopic and should be stored at 2–8 °C in a sealed container under dry nitrogen; for retention beyond 12 months, storage at −20 °C is advised because the solid can cake and undergo hydrolysis after repeated cold-to-ambient cycling.
| Parameter | Typical acceptance | Test method |
|---|---|---|
| Appearance | white to off-white powder | visual inspection |
| Assay | ≥98.0% area | reversed-phase HPLC, 210 nm |
| Water | ≤0.5% | Karl Fischer, USP 921 / Ph Eur 2.5.12 |
| Enantiomeric excess | ≥99.0% | chiral HPLC, Chiralpak ZWIX(+) |
| Chloride content | 18.8–20.2% | titration/ion chromatography |
| Residual solvents | complies USP 467 | headspace GC |
| Storage | 2–8 °C, nitrogen | supplier protocol |
In a production environment with relative humidity above 60%, the powder should be warmed to ambient temperature inside a nitrogen-purged glove bag or dry room before weighing. Direct transfer from cold storage into humid air causes condensation on the crystal surface and creates a sticky film that retards flow through solids-handling equipment. On pilot-scale pressure filters, the hydrochloride salt forms a low-permeability cake; nitrogen-assisted filtration and careful washing with cold methyl tert-butyl ether reduce but do not eliminate the risk of channeling. If the solid develops a yellow cast or persistent gumming on the container wall, the lot should be requalified by HPLC before charging to a reactor. Residual solvent acceptance should be aligned with ICH Q3C classes for the intended use; dichloromethane and methanol are common process solvents and their limits are not uniform across suppliers.
The key difference is the mechanism and selectivity of carboxyl deprotection. The methyl and ethyl esters of L-alanine are smaller and more water-soluble, but their removal requires saponification with alkali or strong aqueous acid hydrolysis, which can hydrolyse N-protecting groups and racemise the α-carbon if pH and temperature are not tightly controlled. The tert-butyl ester is removed by non-aqueous acidolysis, usually trifluoroacetic acid in dichloromethane at 20–25 °C, through an SN1-type fragmentation to the tert-butyl cation and isobutylene. This allows selective C-terminal deprotection while retaining N-terminal Fmoc or other acid-stable carbamate protecting groups. The molecular-weight penalty is also greater for the tert-butyl ester: 181.66 g mol⁻¹ compared with 139.58 g mol⁻¹ for L-alanine methyl ester hydrochloride and 153.61 g mol⁻¹ for L-alanine ethyl ester hydrochloride. Comparative data are given in Table 2.
| Property | L-alanine tert-butyl ester HCl | L-alanine methyl ester HCl | L-alanine ethyl ester HCl |
|---|---|---|---|
| CAS | 13404-22-3 | 2491-20-5 | 1115-59-9 |
| Formula weight | 181.66 g mol⁻¹ | 139.58 g mol⁻¹ | 153.61 g mol⁻¹ |
| Typical deprotection | TFA/DCM, 20–25 °C | aqueous NaOH/THF or HCl/reflux | aqueous NaOH/EtOH or HCl/reflux |
| Carboxyl selectivity | acid-selective; Fmoc retained | low; base removes Fmoc/ester | low; base removes Fmoc/ester |
| Solubility profile | DMF, DCM, methanol; slow hydrolysis in water | water, methanol; faster hydrolysis | water, ethanol; faster hydrolysis |
| Storage | 2–8 °C, dry, nitrogen | 2–8 °C, dry, nitrogen | 2–8 °C, dry, nitrogen |
The tert-butyl ester hydrochloride is also distinct from the corresponding free ester. The free ester is a moderately volatile liquid that is difficult to store and must be prepared immediately before use, whereas the hydrochloride is a stable crystalline solid. The salt is not directly acylated; it must be neutralised with a tertiary base. Piperidine should not be used for this neutralisation because it can deprotect Fmoc groups and may promote α-carbon racemisation in polar aprotic solvents under slow addition. Secondary amines are generally avoided until after acylation.
Compared with benzyl and allyl ester protecting groups, the tert-butyl ester offers acid-mediated deprotection rather than hydrogenolysis or palladium-catalysed cleavage. This distinction is relevant when the substrate contains sulfur- or nitrogen-rich motifs that poison hydrogenation catalysts or when the API intermediate cannot tolerate residual palladium above the permitted limit. Benzyl esters are often removed by catalytic hydrogenation or strong acid, while allyl esters require palladium-catalysed transfer. The tert-butyl ester hydrochloride can be removed under mild non-aqueous conditions without a metal catalyst, which simplifies equipment cleaning and avoids expensive catalyst recovery steps. However, the tert-butyl ester is not suitable for sequences requiring selective C-terminal release in the presence of acid-sensitive side-chain protecting groups such as trityl or Mtt; in such cases the methyl or ethyl ester may be preferred if a final saponification can be tolerated.
In a typical coupling protocol, the hydrochloride is suspended in anhydrous DMF at 0–5 °C and treated with 1.0–1.05 equivalents of N,N-diisopropylethylamine or N-methylmorpholine. The base addition is exothermic; on a 100 L glass-lined reactor with a retreat-curved impeller, the jacket is held at 0 °C and the base is added over 30–45 min to prevent a local temperature spike. A local pH rise above the target can racemise the alanine α-carbon, especially when the coupling reagent is added as a concentrated solution. After neutralisation, the N-protected amino acid and the coupling reagent—usually HATU or EDCl with HOBt—are introduced in sequence, and the batch is monitored by reversed-phase HPLC. Conversion below 95% at 4 h is more commonly caused by residual protonated amine or water ingress than by low reagent reactivity. The C-terminal tert-butyl ester peptide is isolated by extraction into ethyl acetate or methyl tert-butyl ether, washed with dilute citric acid and sodium bicarbonate, and dried over magnesium sulfate. The tert-butyl ester is then removed with TFA/DCM before further chain elongation.
The free amine generated by neutralisation is short-lived in the presence of water or alcoholic co-solvents; slow acylation leads to hydrolysis of the tert-butyl ester and release of L-alanine. The reaction should therefore be started within 15–30 min after complete base addition. In pilot batches, a delayed addition of the activated ester is a common cause of high L-alanine content in the final API intermediate.
Chiral integrity of the product is evaluated by chiral HPLC because specific rotation is not sufficiently sensitive to detect 1% D-enantiomer in a lot. A Chiralpak ZWIX(+) column with methanol/water/formic acid eluent resolves L-alanine tert-butyl ester hydrochloride from the D-enantiomer; the release acceptance is usually ≥99.0% enantiomeric excess. Loss of stereochemical purity during storage is low if the material is kept dry and below 8 °C, but repeated exposure to humid air can produce local acidic films that promote both hydrolysis and racemisation. In coupling campaigns, the stereochemical outcome is confirmed by conversion to a diastereomeric amide and analysis by reversed-phase HPLC rather than relying on optical rotation alone.
When the tert-butyl ester is removed, the peptide solution is typically treated with a TFA/DCM mixture at 20–25 °C. Isobutylene is evolved and should be scrubbed or directed to a cold trap because it is flammable. After 1–3 h, the reaction is concentrated under reduced pressure below 35 °C; higher pot temperatures cause partial cleavage of acid-sensitive side-chain protecting groups and discolouration. The resulting C-terminal carboxylic acid may be isolated as a TFA salt or neutralised immediately before the next coupling. Published data for continuous-flow deprotection of this specific hydrochloride is limited, so batch studies with HPLC monitoring are recommended.
When the hydrochloride salt is used in large-scale peptide manufacture, the main process constraint is deprotection mass intensity rather than coupling selectivity. Cleavage of the tert-butyl ester generates isobutylene and TFA-derived by-products; the waste stream is usually neutralised with aqueous base and sent to solvent recovery. The atom-efficiency penalty is real: 181.66 g of the hydrochloride salt supplies the same alanine residue as 139.58 g of the methyl ester hydrochloride, and the deprotection step consumes TFA far in excess of the stoichiometric requirement because TFA is usually used as a co-solvent. These factors are acceptable only when the acid-selective removal of the tert-butyl group simplifies separation or preserves an acid-stable N-protecting group in later steps. The salt is not suitable for aqueous peptide ligation, for direct use in solid-phase synthesis without neutralisation, or for processes where residual chloride is incompatible with the final salt form. It is also incompatible with strongly acidic aqueous media above 40 °C, which causes rapid deprotection and releases L-alanine hydrochloride.
Safety-relevant handling limits follow from the release of hydrogen chloride and isobutylene. The solid itself has a low vapour pressure, but dust generated during transfer should be controlled by local exhaust ventilation. Contact with damp skin releases hydrogen chloride and forms a sticky film; nitrile gloves and safety goggles are standard. During scale-up, any equipment opened after storage under nitrogen should be treated as oxygen-deficient until purged. Spillage is collected dry and transferred to a closed waste container; water washdown is avoided because it converts the ester to alanine hydrochloride and produces isobutylene.
For identity confirmation, 1H NMR in DMSO-d6 shows the tert-butyl singlet near 1.45–1.50 ppm and the alanine methine near 3.85–3.95 ppm; the hydrochloride proton appears as broad exchangeable absorption. The 13C NMR carbonyl resonance for the tert-butyl ester is observed near 168–170 ppm. Exact shifts vary with field and referencing and are not release criteria. Mass spectrometric confirmation is by electrospray ionisation; the protonated free base [M+H]+ is observed at 146.1 m/z for the free ester, while the hydrochloride salt can show chloride adducts in negative-ion mode. These data may be included in the certificate of analysis when identity by NMR alone is not sufficient for customs or regulatory review.
