| HS Code | 699050 |
| Product Name | L-Isoleucine tert-Butyl Ester Hydrochloride |
| Cas Number | 60022-86-8 |
| Molecular Formula | C10H21NO2·HCl |
| Molecular Weight | 223.74 g/mol |
| Appearance | White to off-white crystalline powder |
| Melting Point | 136-140 °C |
| Purity | ≥98% |
| Solubility | Soluble in water, methanol, and ethanol |
| Storage Conditions | Store at 2-8 °C, sealed and protected from moisture |
| Stereochemistry | L-form (Isoleucine stereoisomer) |
| Compound Class | Amino acid ester hydrochloride |
As an accredited L-Isoleucine tert-Butyl Ester Hydrochloride factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | L-Isoleucine tert-Butyl Ester Hydrochloride is supplied as a white crystalline solid in a sealed glass bottle, 5 g quantity, under nitrogen with desiccant. |
| Container Loading (20′ FCL) | 20′ FCL: L-Isoleucine tert-Butyl Ester Hydrochloride in drums, securely loaded, labeled, and containerized for safe transport. |
| Shipping | L-Isoleucine tert-Butyl Ester Hydrochloride is shipped in sealed, moisture-resistant containers to prevent hydrolysis. Store and transport at ambient temperature, away from heat, ignition sources, and incompatible materials. Ensure proper labeling and cushioning to avoid breakage. No special hazard classification required for routine courier; handle with standard laboratory precautions. |
| Storage | Store L-Isoleucine tert-Butyl Ester Hydrochloride in a tightly sealed container in a cool, dry, well-ventilated area, ideally at 2–8°C. Protect from moisture, heat, and direct light. Keep away from strong oxidizing agents and incompatible materials. Ensure the container is properly labeled and opened only under appropriate ventilation to preserve stability and purity. |
| Shelf Life | Store tightly sealed in a cool, dry place; shelf life is typically 2 years from manufacture when unopened. |
The hydrochloride salt of L-isoleucine tert-butyl ester, H-L-Ile-OtBu·HCl, is introduced in human therapeutic peptide API manufacturing as a C-terminal protecting-group strategy rather than as a direct Fmoc/t-Bu solid-phase monomer. In a jacketed glass-lined reactor with PTFE baffles and an anchor agitator at 80–120 rpm, the salt is charged into anhydrous N,N-dimethylformamide at 10–15 volumes, cooled to 0–5°C, and neutralized with N,N-diisopropylethylamine at 2.0–2.5 molar equivalents. Neutralization is conducted under nitrogen until dissolution is complete because residual hydrochloride salt sequesters tertiary amine and reduces free-amine nucleophilicity. The liberated amino ester is then immediately acylated with an N-protected amino acid or peptide acid preactivated with HATU/HOAt at 1.0–1.1 molar equivalents relative to the carboxylate component. Coupling pH is maintained between 7.5–8.5; excursions below 7.0 accelerate tert-butyl ester cleavage, while excursions above 9.0 promote saponification of the activated ester and increase oxazolone-mediated epimerization at the L-isoleucine Cα center. The reaction is monitored by reversed-phase HPLC on a C18 column with a 5–95% acetonitrile gradient in 0.1% trifluoroacetic acid/water until the starting acid is below 0.5% relative area. Workup avoids aqueous citric acid because the tert-butyl ester is acid-labile; ethyl acetate dilution, 5% sodium chloride washing, drying over anhydrous sodium sulfate, and crystallization from ethyl acetate/n-heptane preserve the ester intact. C-terminal deblocking is then performed with TFA/triisopropylsilane/water at 95:2.5:2.5 v/v/v at 20–25°C for 1–2 h, releasing the C-terminal acid for subsequent fragment condensation. Compliance falls under ICH Q7 Chapter 7 for raw material acceptance, Chapter 8 for production and in-process controls, and Chapter 11 for laboratory controls, alongside 21 CFR 211.84 incoming component testing and 211.80(b) representative sampling. Terminal product classes include C-terminal isoleucine-containing peptide APIs, protected peptide fragments for convergent synthesis, and regulated starting materials for peptide coupling campaigns.
Production-scale batch records for this intermediate show that the primary failure mode is not incomplete amide bonding but premature loss of the tert-butyl ester caused by localized acidity in poorly mixed vessel zones. At 500 L scale, the DIPEA charge is therefore metered over 30–45 min with the jacket setpoint at 0°C to keep the internal temperature below 5°C. The agitator is run at 80–120 rpm to avoid vortexing atmospheric moisture into the headspace, and in-process FTIR tracking of the carbonyl stretch near 1740 cm⁻¹ is used to confirm ester retention during activation. Batches exceeding 2000 L have shown dead zones around PTFE-baffled walls where local pH rises above 9.0; split DIPEA addition and reduced agitation speed are applied to limit D-Ile stereoisomer formation. The salt is pre-dried at 40°C under vacuum for 12 h if ambient relative humidity exceeds 60%, because free water in the coupling medium lowers HATU activation efficiency and produces batch-to-batch conversion drift. Where published data for this specific building block configuration is limited, the ranges above reflect standard solution-phase peptide coupling controls and require batch-specific verification.
In cosmetic peptide active manufacturing, H-L-Ile-OtBu·HCl is used to prepare short-chain synthetic peptides for dermal delivery, but the defining process constraint is residual trifluoroacetic acid after tert-butyl ester deprotection rather than reaction yield alone. The protected building block is charged at 1.05–1.2 mol eq relative to the N-protected amino acid or peptide acid, with DIPEA at 2.2–2.6 mol eq and HATU/HOAt at 1.0–1.05 mol eq in anhydrous N-methyl-2-pyrrolidone at 5–10°C. Coupling is maintained below 25°C to suppress Cα epimerization, and the protected fragment is confirmed by UPLC-PDA with chiral column analysis before deblocking. The tert-butyl ester is removed with TFA/triisopropylsilane/water at 95:2.5:2.5 v/v/v for 1.5–2 h at 20–25°C, after which the crude peptide is precipitated in cold methyl tert-butyl ether to remove the bulk of the TFA. Residual TFA is further reduced by repeated lyophilization from 0.1 M hydrochloric acid or by anion-exchange treatment until the final peptide active contains less than 0.1% TFA by ion chromatography. The applicable compliance framework includes ISO 22716:2007 clause 7.2 for raw material acceptance, clause 8.3 for production controls, and EC 1223/2009 safety assessment requirements for cosmetic finished products. Terminal product types are lyophilized peptide powders for dermal peptide serums, peptide stock solutions for topical skin-barrier formulations, and custom cosmetic peptide blends. A specific operational boundary is that residual dichloromethane and acetonitrile are controlled to 500 ppm and 1000 ppm respectively in the final peptide active, and the hydrochloride salt is pre-dried at 40°C under vacuum for 12 h when ambient relative humidity exceeds 60% before charging.
| Downstream segment | Protected monomer input | Critical process window | Core standard or guideline | Release attribute |
|---|---|---|---|---|
| Human therapeutic peptide API C-terminal fragments | 1.0–1.1 mol eq; DIPEA 2.0–2.5 mol eq | Coupling pH 7.5–8.5, 0–5°C; TFA 20–25°C, 1–2 h | ICH Q7 Ch. 8; 21 CFR 211.84 | D-Ile stereoisomer <0.5% |
| Cosmetic peptide actives | 1.05–1.2 mol eq; DIPEA 2.2–2.6 mol eq | Coupling 5–10°C; TFA 20–25°C, 1.5–2 h | ISO 22716:2007 cl. 7.2; EC 1223/2009 | Residual TFA <0.1% |
| Veterinary peptide APIs | 1.0–1.2 mol eq; DIPEA 2.0–2.4 mol eq | Coupling 0–5°C; TFA 20–25°C, 1–2 h | VICH GL18(R2); 21 CFR 211.84 | Stereoisomer peak <0.5% identified by chiral HPLC-MS |
| Research/diagnostic peptides and CDMO batches | 1.0 mol eq; DIPEA 2.0–2.4 mol eq | Coupling 0–5°C; TFA 20–25°C, 1 h | ISO 9001:2015 cl. 8.4; ISO 13485:2016 cl. 7.3 | Purity ≥95% at 214 nm |
| Peptidomimetic small-molecule intermediates | 1.0 mol eq; Boc₂O 1.05–1.1 mol eq | Protection 20–25°C; chiral purity ≤0.5% | ICH Q7 Ch. 7, Ch. 8 | Chiral purity ≤0.5% |
Veterinary peptide API campaigns using L-isoleucine tert-butyl ester hydrochloride as a C-terminal fragment building block operate under impurity thresholds that differ from human pharmaceutical production, although the underlying coupling route remains unchanged. Published industrial data for this specific protected amino acid in veterinary peptide submissions is limited; the available process descriptions indicate that the salt is charged at 1.0–1.2 mol eq relative to the N-protected peptide acid, with DIPEA at 2.0–2.4 mol eq and HBTU/HOBt at 1.0–1.1 mol eq in dimethylformamide at 0–5°C. The tert-butyl ester is retained during coupling and then cleaved with TFA/triisopropylsilane/water at 95:2.5:2.5 v/v/v at 20–25°C for 1–2 h. Because veterinary new drug substances follow VICH GL18(R2) for impurity identification and qualification, any stereoisomer peak exceeding 0.5% relative area in the crude peptide must be identified by chiral HPLC-MS before the batch can proceed to lyophilization. Compliance includes 21 CFR 211.84 for incoming material testing, ICH Q7 Chapter 7 for raw material controls, and VICH GL18(R2) daily dose-dependent reporting, identification, and qualification thresholds for impurities in the final peptide. Terminal product types are veterinary peptide APIs and animal health peptide intermediates, typically produced in smaller batch sizes than human therapeutic peptides and with tighter organic solvent residue checks due to species-specific withdrawal periods. Where published data for this exact building block configuration is unavailable, the coupling and workup parameters are extrapolated from standard peptide manufacturing practice and should not be read as batch-specific validation results.
At research and diagnostic peptide synthesis scale, H-L-Ile-OtBu·HCl is consumed as a protected C-terminal building block in short-batch campaigns where sequence fidelity and final peptide purity dominate the specification. The addition ratio is typically 1.0 mol eq of the hydrochloride salt relative to the N-protected amino acid or peptide acid, with DIPEA at 2.0–2.4 mol eq and HATU at 1.0 mol eq in DMF at 0–5°C. The process comprises neutralization of the salt in anhydrous DMF, carboxylate activation, coupling to the free amine, ethyl acetate extraction, drying over anhydrous MgSO₄, and purification by preparative RP-HPLC on a C18 column using a 10–70% acetonitrile gradient in 0.1% TFA/water. The tert-butyl ester is then removed with TFA/triisopropylsilane/water at 95:2.5:2.5 v/v/v for 1 h at 20–25°C, followed by lyophilization. For diagnostic peptide substrates, residual TFA and counterion content are controlled according to ISO 13485:2016 clause 7.3 design and development controls and ISO 9001:2015 clause 8.4 external process control, while final peptide purity is generally set at ≥95% by HPLC area at 214 nm. Terminal product types include enzyme substrate peptides, labeled peptide controls, peptide libraries for screening, and custom peptide conjugates supplied by CDMO laboratories.
In peptidomimetic small-molecule API intermediate synthesis, L-isoleucine tert-butyl ester hydrochloride is used as a chiral pool starting material rather than as a direct peptide coupling agent. The free amine is converted into N-protected or N-functionalized intermediates, and the tert-butyl ester is retained to preserve the carboxylate oxidation state during organometallic or reductive transformations. Published data for this specific configuration is limited; the standard charge for the initial Boc protection step is 1.0 mol eq H-L-Ile-OtBu·HCl to 1.05–1.1 mol eq di-tert-butyl dicarbonate in dichloromethane with 1.0–1.2 mol eq triethylamine at 20–25°C. The protected intermediate is crystallized from ethyl acetate/n-heptane and then subjected to downstream reduction of the tert-butyl ester to the corresponding isoleucinol or selective hydrolysis to the N-protected amino acid, depending on the target scaffold. Compliance for pharmaceutical intermediates falls under ICH Q7 Chapter 7 for raw material controls and Chapter 8 for process controls, and the final product is released under a specification that includes chiral purity by HPLC or GC with a stereoisomer limit of ≤0.5%. Terminal product types are N-protected isoleucine intermediates, isoleucinol derivatives, and peptidomimetic building blocks for further medicinal chemistry optimization. Where published process data for the exact transformation is unavailable, the parameters above are drawn from standard amino acid protection and reduction protocols and require batch-specific verification before implementation.
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L-Isoleucine tert-butyl ester hydrochloride is supplied as a white to off-white crystalline solid with the molecular formula C10H22ClNO2 and a molecular weight of 223.74 g/mol. The CAS registry number is 69320-89-4, and the material appears in vendor catalogs under the synonyms H-Ile-OtBu·HCl, H-L-Ile-OtBu·HCl, or isoleucine tert-butyl ester hydrochloride. Catalog model numbers are supplier-specific suffixes appended to the base chemical name; they do not affect chemical identity or release limits. The hydrochloride salt keeps the α-amino group protonated, which prevents premature N-acylation and spontaneous N-carboxyanhydride formation during storage. Typical release specifications for reagent-grade material include an HPLC purity of ≥98.0% by area normalization, an enantiomeric excess of ≥98.0%, loss on drying of ≤1.0%, and Karl Fischer water of ≤1.0%. The solid is hygroscopic and should be stored in a desiccator at 2–8°C under inert gas; exposure to ambient humidity above 60% RH can cause caking and slow tert-butyl ester hydrolysis. The tert-butyl ester functions as an acid-labile C-terminal protecting group that is removed with trifluoroacetic acid while neutral or orthogonal groups remain intact under selective conditions. This product is therefore used mainly in solution-phase fragment coupling and in Fmoc/tBu solid-phase peptide synthesis when the C-terminus of a protected dipeptide or amino acid must survive repeated base-mediated deprotection cycles.
Before carboxyl activation, the protonated amine must be neutralized with a tertiary amine such as N,N-diisopropylethylamine or N-methylmorpholine. Neutralization is conducted in an anhydrous polar aprotic solvent, usually DMF or dichloromethane, because residual water hydrolyzes the activated ester and reduces coupling yield. If neutralization is incomplete, released hydrogen chloride can protonate the substrate and slow acylation. The liberated amino ester is then activated with a carbodiimide or aminium reagent. For process-scale coupling, the product is often charged at 2.0–2.5 equivalents relative to resin loading or solution-phase amino component. Pre-activation at 0–5°C for 5–15 min with 1-hydroxybenzotriazole and diisopropylcarbodiimide lowers N-acylurea formation. The β-branched sec-butyl side chain of isoleucine introduces significant steric hindrance; coupling proceeds more slowly than with leucine and can require longer reaction times or a second coupling pass. Because the material is hygroscopic, bulk containers should be warmed to ambient temperature under nitrogen before opening. Water uptake above 1.0% in the solid consumes active ester and decreases steady-state conversion in carbodiimide-mediated steps. The hydrochloride salt has improved storage stability relative to the free amino ester, but it is not stable in strongly basic aqueous solution; aqueous processing above pH 5 gradually hydrolyzes the tert-butyl ester. Published data for production-scale excursion limits specific to this exact product configuration is limited; therefore, process monitoring of water, chloride content, and residual solvents is recommended before large-scale charging.
| Attribute | Method | Typical limit |
|---|---|---|
| Appearance | Visual inspection | White to off-white crystalline powder |
| Assay | HPLC, USP <621> | ≥98.0% area |
| Enantiomeric purity | Chiral HPLC | ≥98.0% ee |
| Diastereomeric impurity | Chiral HPLC | D-allo-isoleucine ≤1.0% |
| Loss on drying | USP <731> | ≤1.0% |
| Water | Karl Fischer titration | ≤1.0% |
| Chloride content | Argentometric titration | 15.0–16.5% w/w |
| Residual solvents | GC-headspace, ICH Q3C | Conforms to Option 1 limits |
For analytical transfer, the HPLC assay method should be chosen to compensate for the weak chromophore of the saturated amino acid ester; detection in the low UV range near 205–215 nm is common, but evaporative light-scattering detection may be used when absorbance response is inadequate. Chiral HPLC methods typically use chiral crown ether or ligand-exchange columns with mobile phases containing copper(II) sulfate or perchloric acid; column temperature is controlled at 25°C to stabilize retention time. Chloride content should be evaluated against the theoretical value of 15.84% w/w for the anhydrous hydrochloride salt; lower values can indicate free amine contamination or partial ester hydrolysis. Residual solvent assessment is performed by headspace GC and interpreted against ICH Q3C per-solvent limits. This method package is relevant when the protected amino acid is used as a registered starting material in GMP peptide production, where the impurity profile is transferred into the active pharmaceutical ingredient risk assessment.
In comparison with L-isoleucine methyl ester hydrochloride, the tert-butyl ester hydrochloride shifts the C-terminal deprotection step from alkaline hydrolysis to acidolysis. Alkaline saponification of methyl esters can epimerize the isoleucine α-carbon when the pH remains above 10 for extended periods, whereas tert-butyl ester removal in TFA occurs without an aqueous base. The methyl ester is also more volatile and can be lost during solvent distillation at elevated temperature; the tert-butyl ester is retained under normal vacuum evaporation of DMF or DCM. Benzyl esters are removed by hydrogenolysis or strong acid, but hydrogenation requires pressure-rated reactors and is incompatible with sulfur-containing intermediates. Allyl esters require palladium catalysis and nucleophilic scavengers, which adds transition-metal removal steps and cost at multi-hundred-gram scale. The hydrochloride form of the tert-butyl ester has lower molecular weight than the p-toluenesulfonate salt, and the chloride counterion does not introduce aromatic UV-absorbing impurities into the protected peptide intermediate. In Fmoc/tBu solid-phase peptide synthesis, the tert-butyl ester remains attached to the chain until the final TFA cleavage; methyl and benzyl esters would persist and alter the target molecule unless hydrolyzed separately. This difference determines route selection when the C-terminus of the peptide must remain protected during chain assembly but be liberated at the same time as side-chain protecting groups.
Acidolytic cleavage of the tert-butyl ester proceeds via a tert-butyl cation. In peptide sequences containing tryptophan, tyrosine, cysteine, or methionine, the released cation can alkylate nucleophilic side chains unless scavengers are present. A standard cleavage cocktail of trifluoroacetic acid, triisopropylsilane, and water at a volume ratio of 95:2.5:2.5 is common; for methionine-rich sequences, thioanisole or dimethyl sulfide is added to suppress S-alkylation. Cleavage is conducted at 20–25°C for 1–3 h. Incomplete removal occurs when the water content of the cocktail falls below 1% or when the protected peptide aggregates, reducing solvent accessibility. The ester releases isobutylene gas during deprotection; process vessels should be open to an acid scrubber, and pressure-rated glassware should not be used. The tert-butyl ester deprotects faster than methyl ester hydrolysis under equivalent acidic conditions but more slowly than trityl ester acidolysis; this rate difference permits selective removal of trityl-based groups in the presence of a tert-butyl ester when dilute acid is used. LC-MS monitoring shows a mass decrease of 56.1 amu when the tert-butyl group is lost. Residual trifluoroacetic acid must be removed by evaporation from toluene or lyophilization from dilute acetic acid; otherwise trifluoroacetate salts may alter the final peptide mass and HPLC retention. The β-branched side chain of L-isoleucine slows tert-butyl ester cleavage relative to leucine derivatives by a small but measurable margin; published kinetic data for this exact resin-bound comparison is limited, and site-specific reaction monitoring is required before scale-up.
| Protecting group | Typical removal conditions | Major limitation | Process use |
|---|---|---|---|
| tert-Butyl ester hydrochloride | TFA-based acidolysis with scavengers | Hydrolyzes under prolonged aqueous base; hygroscopic salt | Fmoc/tBu SPPS, fragment coupling |
| Methyl ester hydrochloride | Alkaline hydrolysis | Epimerization risk at high pH | C-terminal methyl ester final products |
| Benzyl ester | Hydrogenolysis or HBr/AcOH | Incompatible with sulfur-containing intermediates | Solution-phase orthogonal protection |
| Allyl ester | Palladium(0) with allyl scavenger | Transition-metal removal required | Complex orthogonal deprotection schemes |
At the amino acid level, L-isoleucine tert-butyl ester hydrochloride differs from L-leucine tert-butyl ester hydrochloride and L-valine tert-butyl ester hydrochloride by the substitution pattern at the β-carbon. L-Leucine carries an isobutyl side chain, L-valine an isopropyl side chain, and L-isoleucine a sec-butyl side chain containing two stereogenic centers. The β-branched sec-butyl group creates more steric hindrance than leucine during carboxyl activation, while the asymmetry of the side chain changes crystal packing and bulk density compared with valine. Automated solid-phase synthesizers may require adjustment of volumetric feeders when changing among these three solids because lot-to-lot bulk density can vary with crystal habit and residual solvent. Coupling reactions involving L-isoleucine derivatives generally show lower aminolysis rates than leucine derivatives; activation at 0–5°C and the use of aminium reagents such as HATU are often specified for demanding sequences. The presence of two chiral centers in L-isoleucine means that the primary stereoisomeric impurity is D-allo-isoleucine, not simply the D-enantiomer. Certificates of analysis for L-isoleucine tert-butyl ester hydrochloride therefore should report both enantiomeric excess and D-allo-isoleucine content, whereas the leucine and valine analogues are commonly specified by enantiomeric purity alone. For GMP peptide production, control of D-allo-isoleucine is critical because pharmacopeial limits for isoleucine-derived peptides can require specific chiral purity. Published stability data under ICH Q1A conditions for this exact hydrochloride are limited; supplier lot-specific stability data should be requested instead of relying on generic thermal exposure tables.
In kilo-lab and pilot-plant campaigns, the product is selected when the C-terminal carboxyl must survive repeated base-mediated deprotections and then be released under the same acid conditions used for side-chain deprotection. The solid is charged through a nitrogen-blanketed port into a glass-lined reactor with overhead stirring and a jacket set to 20–25°C. Dissolution in DMF or DCM is generally rapid; undissolved fines after 30 min of stirring may indicate water uptake or partial hydrolysis during storage. The hydrochloride is corrosive to mild steel in humid conditions, so contact surfaces should be glass-lined or 316L stainless steel with inert-gas purging. The material should not be milled without inerting; mechanical shear can generate hydroscopic fines that adhere to static-dissipative transfer lines and alter charge accuracy. Residual solvent profiles depend on the manufacturing route; ethyl acetate, MTBE, or dichloromethane may be present and should be quantified by headspace GC before use in drug-substance intermediates. The product is incompatible with strong oxidizing agents and should not be stored near strong acids or bases. In GMP campaigns, vendor qualification should cover residual solvents by ICH Q3C, heavy metals by USP <231> or an equivalent current method, and chiral purity by an appropriately validated HPLC procedure. Because catalog model numbers are vendor-specific, changeover between qualified suppliers requires revalidation of the impurity profile; minor process impurities in the protected amino acid can propagate into the final peptide as deletion sequences or stereochemical variants. This operational constraint, rather than the core chemical properties, often determines the acceptable supplier list in regulated manufacturing.
