| HS Code | 591864 |
| Product Name | BOC-L-threonine |
| Iupac Name | (2S,3R)-2-[(tert-butoxycarbonyl)amino]-3-hydroxybutanoic acid |
| Cas Number | 2592-18-9 |
| Molecular Formula | C9H17NO5 |
| Molecular Weight | 219.24 g/mol |
| Appearance | White to off-white crystalline powder |
| Melting Point | 82-85 °C |
| Optical Rotation | -6.5° (c=1 in methanol) |
| Purity | ≥98.0% |
| Solubility | Soluble in methanol, ethanol, DMF, and DMSO; sparingly soluble in water |
| Storage Condition | Store at 2-8 °C, tightly sealed, protected from moisture |
| Pka | 3.78 |
As an accredited BOC-L-threonine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | BOC-L-threonine, 25 g, supplied in a sealed amber glass bottle with tamper-evident cap, labeled with purity and storage conditions. |
| Container Loading (20′ FCL) | 20′ FCL container loading of BOC-L-threonine: securely packed in sealed drums/pails, palletized, ventilated, and properly documented for safe transport. |
| Shipping | BOC-L-threonine ships at ambient temperature in sealed, moisture-resistant containers. Keep away from heat, sparks, and incompatible materials. Store in a cool, dry place after receipt. Avoid dust inhalation and skin contact. Ensure proper labeling and documentation for chemical transport. |
| Storage | Store BOC-L-threonine in a tightly sealed container under inert gas, protected from light and moisture. Recommended storage temperature is 2–8 °C for short-term use, or –20 °C for prolonged stability. Avoid repeated temperature fluctuations. Keep away from heat, acids, bases, and oxidizing agents. Always allow container to warm to room temperature before opening to prevent condensation. Use under dry conditions to maintain purity. |
| Shelf Life | Store sealed in a cool, dry place. Typical shelf life is 2–3 years from manufacture when handled properly. |
Automated batch solid-phase peptide synthesis using tert-butoxycarbonyl chemistry constitutes an established manufacturing route for short-chain peptide active pharmaceutical ingredients in which L-threonine occupies a position in the primary sequence. In this route, BOC-L-threonine is dissolved in a DMF/DCM mixed solvent system at concentrations between 0.3 M and 0.6 M and charged at 2.5–4.0 molar equivalents relative to the free amine loading of aminomethyl polystyrene or 4-methylbenzhydrylamine resin with initial substitution levels of 0.5–1.0 mmol/g. The process is operated under ICH Q7 and ICH Q11 guidance, with residual solvent release testing aligned to ICH Q3C and USP General Chapter <467>, and elemental impurity control aligned to ICH Q3D and USP General Chapter <232>/<233>. Coupling is performed with HBTU or TBTU in the presence of HOBt and DIPEA, using an activation interval of 2–15 min and a coupling recirculation interval of 30–90 min in a 500 L glass-lined SPPS reactor with a 316L stainless steel sintered filter bottom and nitrogen sparge. The N-terminal tert-butoxycarbonyl group is removed with 20–40% TFA in DCM over 10–30 min, followed by neutralization with 5% DIPEA in DMF. Because BOC-L-threonine carries an unprotected side-chain hydroxyl, prolonged activation or excess acylating reagent generates O-acylated threonine adducts; batch records therefore restrict pre-activation time and prescribe acetic anhydride/pyridine capping after each coupling cycle. Final peptide is cleaved with liquid HF or trifluoromethanesulfonic acid in the presence of anisole and dimethyl sulfide at 0°C for 1–2 h, precipitated with chilled MTBE, and purified by preparative RP-HPLC on C8 or C18 columns using acetonitrile and 0.1% TFA gradients. Terminal finished products are compendial peptide APIs containing L-threonine residues, including somatostatin analogue acetates that require a C-terminal threoninol modification. Published data on campaign-specific yields varies, but the free hydroxyl side chain is a recurring source of synthesis-related impurity formation when the validated activation window shifts by more than 5 min.
Solution-phase fragment condensation of BOC-L-threonine is selected for short protected peptide fragments when resin-based manufacturing is not economically justified or when a specific activated fragment is required for regioselective coupling. The protected monomer is charged at 1.0–1.2 molar equivalents relative to a carboxyl-activated peptide or amino acid fragment. Activation proceeds through mixed anhydride formation with isobutyl chloroformate and N-methylmorpholine in THF/DMF at temperatures between −15°C and −10°C, with coupling maintained for 6–12 h. The downstream unit operations include aqueous workup with 10% citric acid and 5% sodium bicarbonate, solvent distillation under reduced pressure below 35°C, and crystallization from MTBE/heptane mixtures to a chromatographic purity above 99.0% by area percent. Quality obligations for early-phase fragment production are assigned under ISO 9001:2015; if the fragment is forwarded into a registered API synthesis, ICH Q7 and ICH Q3C apply to all consecutive unit operations. The terminal products in this route are protected dipeptide, tripeptide, or hexapeptide fragments intended for downstream fragment condensation, not final dosage-form APIs. Residual water in the activation mixture is controlled to less than 0.05% by Karl Fischer titration because the unprotected threonine hydroxyl increases the sensitivity of the mixed anhydride to premature hydrolysis.
| Parameter | Solid-phase Boc-SPPS | Solution-phase fragment condensation |
|---|---|---|
| Molar excess of BOC-L-threonine relative to substrate | 2.5–4.0 molar equivalents | 1.0–1.2 molar equivalents |
| Solvent system | DMF/DCM mixed solvent | THF/DMF mixed solvent |
| Activation chemistry | HBTU or TBTU with HOBt and DIPEA | Isobutyl chloroformate with N-methylmorpholine |
| Reaction temperature | 20–40°C | −15 to −10°C |
| Monitoring method | Kaiser test and quantitative HPLC | TLC and HPLC area-percent |
| Critical side reaction | O-acylation at the free threonine hydroxyl | Hydrolysis of activated carboxylate due to residual water |
BOC-L-threonine is utilized in bench-scale and parallel synthesis of synthetic peptide substrates for protein kinase activity assays because the unprotected hydroxyl retains the phosphorylation-relevant functionality of the native peptide sequence. In this in vitro diagnostic reagent segment, the building block is coupled at 1.1–1.5 molar equivalents relative to resin loading with DIC and HOBt activation in an automated peptide synthesizer fitted with disposable 10 mL reaction vessels. The downstream production process comprises Boc-SPPS chain assembly, TFA cleavage, preparative RP-HPLC at 25–40°C, and lyophilization to a water content below 5%. The applicable quality system is ISO 13485:2016 for in vitro diagnostic component manufacturing, with analytical release testing using USP General Chapter <621> for chromatographic system suitability and USP General Chapter <921> for water determination. Terminal finished products are lyophilized kinase substrate peptides, phosphopeptide calibration standards, and peptide components supplied for assay development kits. The free threonine hydroxyl must be protected from O-acylation during chain assembly; if O-acylation is not controlled, the resulting phosphorylatable impurity co-elutes with the target substrate and reduces assay specificity.
In early-stage peptide drug discovery, BOC-L-threonine is incorporated into synthetic peptide libraries in which threonine is deliberately positioned at solvent-exposed residues to probe target binding, phosphorylation, and metabolic stability. Parallel library synthesis is conducted in 96-well PTFE fritted plates at a 0.1 mmol scale per well, with the amino acid dispensed as a 0.5 M stock solution and charged at 3.0 molar equivalents relative to resin loading. The downstream process uses DIC/HOBt activation in NMP, TFA/TIS/water 95:2.5:2.5 v/v cleavage, and LC-MS purity screening with a release threshold of ≥85%. Documentation is maintained under ISO 9001:2015, and material safety data is aligned with REACH Regulation (EC) No 1907/2006 for research-grade chemical supply. Terminal finished products are research-grade peptide libraries, structure-activity relationship sets, and hit-to-lead peptide candidates. Published data on BOC-L-threonine-specific coupling efficiency across parallel formats is limited; therefore plate-level HPLC sampling is required to detect occlusion of the free hydroxyl during high-throughput activation.
During technology transfer of a threonine-containing somatostatin analog synthesis from pilot to production scale, the coupling behavior of BOC-L-threonine is revalidated because residual water and solvent mischarging in large reactors alter the activation rate of uranium-based coupling reagents. In a 500 L glass-lined SPPS reactor with 316L stainless steel sintered filter bottom, the monomer is charged at 2.5–3.5 molar equivalents relative to resin loading, and the DMF/DCM ratio is held within ±5% of the qualified value. Process analytical technology, typically ReactIR monitoring of carbodiimide-derived intermediate accumulation and in-process HPLC quantification of residual free amine, provides real-time control of the coupling endpoint. The production process includes three sequential steps: N-terminal Boc deprotection with TFA in DCM, neutralization with DIPEA, and carbodiimide/uronium-mediated coupling. Failure modes observed in multi-kilogram campaigns include incomplete neutralization leading to reduced coupling efficiency and O-acylation at the threonine hydroxyl when pre-activation exceeds 10 min. Compliance is maintained under ICH Q7, ICH Q11, and ICH Q8 quality-by-design principles, with impurity fate and purge studies aligned to ICH M7 for potentially mutagenic byproducts. Terminal finished products are multi-kilogram peptide API intermediates or registered starting materials intended for downstream oxidative cyclization and salt formation.
For peptide APIs that require a reduced C-terminal amino alcohol, BOC-L-threonine is used as the starting building block to prepare threoninol-functionalized supports rather than being added as an internal chain-extension monomer. The compound is reduced to N-tert-butoxycarbonyl-L-threoninol via carbodiimide-assisted esterification followed by sodium borohydride reduction, or through borane-THF reduction protocols; the resulting amino alcohol is anchored through its primary hydroxyl to a chlorotrityl chloride resin at a typical loading of 0.4–0.8 mmol/g. The downstream solid-phase chain assembly then uses standard Boc-SPPS cycles: TFA deprotection, DIPEA neutralization, uranium-based coupling, and capping. Compliance for the modified building block is governed by ICH Q7 when the support is used in registered API synthesis, with residual solvent control under ICH Q3C and USP General Chapter <467>. Terminal finished products are C-terminal threoninol-modified peptide APIs, most notably somatostatin analog acetates, in which the threoninol residue is required for receptor binding stability. Published data for this specific BOC-L-threonine reduction and resin loading configuration is limited; batch-dependent aldehyde by-product formation has been observed during uncontrolled borohydride addition and requires chromatographic purging before solid-phase loading.
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BOC-L-threonine (CAS 2592-18-9; molecular formula C9H17NO5; molecular mass 219.24 g/mol) is a tert-butoxycarbonyl-protected L-threonine derivative used as a carboxyl-free N-terminal building block in solid-phase and solution-phase peptide assembly. The product is described by the synonym Boc-Thr-OH and retains the (2S,3R) stereochemistry of L-threonine; the tert-butoxycarbonyl group is attached to the α-amino nitrogen, while the β-hydroxy side chain is not protected. Commercial product models are generally differentiated as peptide synthesis grade and high-purity grade, with the latter subjected to additional enantiomeric purity and residual solvent controls. Because the BOC group is acid-labile and the threonine side chain remains available for O-acylation, the product is positioned for use in BOC-strategy peptide synthesizers and in solution-phase fragment condensations where orthogonal C-terminal activation is required.
Release of BOC-L-threonine on production campaigns is controlled by HPLC area normalization, chiral HPLC, coulometric Karl Fischer titration, residue on ignition, and headspace gas chromatography. A standard peptide synthesis grade is routinely specified at ≥98.0% HPLC purity by UV detection at 205 nm on a 250 mm × 4.6 mm C18 column with 5 μm particle size. High-purity material is specified at ≥99.5%. Enantiomeric purity is tested on a chiral polysaccharide stationary phase with 150 mm × 4.6 mm column dimensions and a mobile phase composed of n-hexane/2-propanol/trifluoroacetic acid 90:10:0.1; the L-threonine-derived enantiomer peak is required at ≥99.0% for peptide synthesis grade and ≥99.7% for high-purity grade. Water content is measured by coulometric Karl Fischer titration according to ASTM E203-16 or Ph. Eur. 2.5.12, with limits of ≤0.50% and ≤0.20%, respectively. Residual solvents are quantified by headspace gas chromatography according to USP <467> or Ph. Eur. 2.4.24; typical limits are ≤0.10% for dichloromethane and ≤0.05% for ethyl acetate. Certificates of analysis for both grades are typically issued by laboratories accredited to ISO/IEC 17025:2017.
| Parameter | Test method | Peptide synthesis grade | High-purity grade |
|---|---|---|---|
| Appearance | Visual inspection under daylight | White to off-white crystalline powder | White crystalline powder |
| HPLC purity | C18 column, detection at 205 nm | ≥98.0% | ≥99.5% |
| Enantiomeric purity | Chiral polysaccharide CSP | ≥99.0% | ≥99.7% |
| Water content | Karl Fischer, ASTM E203-16 | ≤0.50% | ≤0.20% |
| Residue on ignition | Ph. Eur. 2.4.14 | ≤0.20% | ≤0.05% |
| Residual solvents | Headspace GC, USP <467> | ≤0.10% CH₂Cl₂; ≤0.05% EtOAc | ≤0.05% CH₂Cl₂; ≤0.02% EtOAc |
In vacuum tray dryers, BOC-L-threonine is dried with shelf temperature set at 40°C and pressure ≤ 10 mbar for 12–18 h. If water content exceeds 0.50%, the powder can form soft agglomerates in pneumatic transfer lines, producing feed variability at automated resin-loading stations. The compound is handled in low-humidity environments at RH < 30% to preserve free-flowing behavior on double-screw gravimetric feeders. Redrying at 45°C or higher is not recommended because the tert-butoxycarbonyl group can undergo thermal deprotection with release of isobutylene and carbon dioxide; differential scanning calorimetry on 10 mg samples at 10°C/min heating rate typically shows a decomposition endotherm near 80–85°C. Published data for this specific configuration is limited, so thermal conditions above 40°C should be validated by lot-specific purity assay before use.
BOC-L-threonine is activated in solution-phase syntheses with N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in combination with 1-hydroxybenzotriazole hydrate (HOBt·H₂O) or Oxyma Pure. The preferred activation protocol uses 1.0–1.2 equiv of the carbodiimide and 1.0–1.2 equiv of the additive relative to BOC-L-threonine in dimethylformamide at 0–4°C. N,N-diisopropylethylamine is added at 2.0–2.5 equiv while maintaining internal temperature below 10°C; base addition is exothermic and requires jacket cooling in 50 L glass reactors. Activation times exceeding 10 min before addition of the amine nucleophile increase the probability of oxazolone formation and α-carbon racemization; the resulting diastereomeric impurities are resolved by chiral HPLC with a 150 mm × 4.6 mm polysaccharide column. In solid-phase mode, a coupling cycle with 2.0 equiv of activated BOC-L-threonine per resin-bound amine, resin substitution 0.5–1.0 mmol/g, and 30–60 min contact time at 20–25°C typically produces a negative Kaiser test result corresponding to residual free amine below 5 μmol/g. The β-hydroxy group can undergo O-acylation when activated amino acid excess is above 3.0 equiv, when DMAP catalysis is used, or when coupling is extended beyond 120 min at 25°C. A capping step with acetic anhydride/pyridine 1:1 v/v for 15 min at 20°C is applied after each coupling to block unreacted resin amine sites.
On automated BOC-strategy peptide synthesizers with reactor volumes of 100 mL, 250 mL, and 2 L, BOC-L-threonine is used without side-chain protection. BOC deprotection is performed with 30–40% trifluoroacetic acid in dichloromethane; the addition of 2–5% v/v anisole or triisopropylsilane quenches the tert-butyl cation and limits alkylation of the peptide chain. TFA-resistant seals, perfluoroelastomer gaskets, and pressure relief lines are required because the deprotection step evolves gaseous by-products. In fragment condensation reactions conducted on 5–10 kg scale, crude protected peptides are precipitated from methyl tert-butyl ether at 0–5°C, collected on nutsche filters with 10–25 μm nominal pore size, and washed until residual TFA is below 0.10% by ion chromatography. The free hydroxyl side chain is compatible with standard ether washing; however, repeated exposure to strongly acidic cleavage mixtures can promote β-elimination at the threonine residue to form α,β-unsaturated C-terminal degradation products.
Switching from Fmoc-L-threonine to BOC-L-threonine changes the protecting group removal regime from base-labile to acid-labile. Fmoc deprotection with 20% piperidine in DMF is fast but introduces a strongly basic environment that can degrade base-sensitive peptide sequences; BOC deprotection with 30–40% TFA in dichloromethane is preferred when the target peptide tolerates acid but not nucleophilic amines. Final peptide cleavage in BOC chemistry typically requires hydrogen fluoride or trifluoromethanesulfonic acid, which demands equipment constructed from polytetrafluoroethylene-lined reactors and fluoropolymer seals; Fmoc chemistry usually uses 95% TFA with scavenger cocktails in glass or polypropylene vessels. The BOC-L-threonine residue has a lower tendency to form diketopiperazines during N-terminal dipeptide assembly than Fmoc analogs under basic conditions, but published data for this specific configuration is limited and the claim should be verified by resin cleavage studies. At multi-kilogram scale, BOC-L-threonine additionally requires acid-handling controls and specialized waste neutralization; process hazard analysis for TFA addition and HF cleavage is mandatory.
| Attribute | BOC-L-threonine | Fmoc-L-threonine | Cbz-L-threonine |
|---|---|---|---|
| N-protecting group removal | 30–40% TFA in DCM | 20% piperidine in DMF | Catalytic hydrogenation over 5% Pd/C |
| Removal mechanism | Acidolysis | Base-mediated elimination | Hydrogenolysis |
| Side-chain status | Free β-hydroxy | Free β-hydroxy or tert-butyl-protected | Free β-hydroxy |
| Final cleavage | HF or TFMSA | TFA/scavenger | HF or hydrogen |
| Main application | BOC SPPS and solution phase | Fmoc SPPS | Solution-phase fragment condensation |
Between BOC-L-threonine and BOC-L-allo-threonine, the principal distinction is the configuration at C-3. BOC-L-threonine is (2S,3R); BOC-L-allo-threonine is (2S,3S). The two compounds are diastereomeric and display different reversed-phase HPLC retention times and different melting ranges. Chiral purity of BOC-L-threonine produced from L-threonine with enantiomeric excess ≥ 99.5% is controlled to prevent contamination with BOC-D-threonine above 0.10%, which would produce peptide diastereomers that may co-elute with the target peptide on C18 columns. Cbz-L-threonine is removed by hydrogenolysis over 5% palladium on carbon at 1–5 bar hydrogen pressure; this route is incompatible with sulfur-containing peptides because sulfur compounds poison the noble metal catalyst. The unprotected β-hydroxy group also distinguishes BOC-L-threonine from BOC-L-threonine methyl ester or tert-butyl ester, in which the carboxyl terminus is masked; the free acid is selected when direct C-terminal activation is needed in solution-phase convergent synthesis.
Storage stability of BOC-L-threonine is influenced by temperature, humidity, and residual solvent composition. Sealed containers stored at 2–8°C under desiccant are used for long-term campaigns; the desiccant should be silica gel or molecular sieve 4A and should be replaced after each opening. Under these conditions, purity retention above 99.0% for 24 months may be reported in vendor stability protocols; however, published data for this specific configuration is limited and site-specific stability studies are recommended. At RH > 60%, uptake of water above 0.50% within 24 h can occur in open containers; this moisture increases the risk of clumping and promotes hydrolytic degradation of the tert-butoxycarbonyl group during storage. Thermal exposure above 40°C accelerates BOC deprotection; storage in non-climate-controlled warehouses should be limited to ≤ 30°C and continuous temperature monitoring with data loggers is recommended. Incompatible storage conditions include close proximity to strong acids, acid chlorides, and amine bases, because both strong acid and strong base can cause deprotection or salt formation. The material is not considered a strong oxidizing agent, but it should be segregated from strong oxidizers to reduce fire risk during waste handling.
During process development, BOC-L-threonine is charged into peptide synthesis campaigns after assay and moisture confirmation. Batch records should include the actual water content and enantiomeric purity because these parameters affect coupling stoichiometry and downstream diastereomer purge. For solid-phase production lots, the resin loading calculation uses molecular mass 219.24 g/mol and the assay value from the certificate of analysis; failure to correct for water or residual solvent content can cause deviations from the targeted resin substitution by up to 1.5% in 2 L reactors. The free β-hydroxy group should be monitored by LC-MS after the first coupling cycle if the target peptide contains base- or acid-labile post-translational modifications. If the HPLC purity of the crude peptide shows a late-eluting impurity near the target peptide retention time, chiral HPLC and high-resolution mass spectrometry are used to determine whether the impurity arises from BOC-D-threonine, O-acylation, or β-elimination.
