CBZ-L-threonine (CAS 19728-63-3), formally N-[(benzyloxy)carbonyl]-L-threonine and abbreviated Cbz-Thr-OH or Z-Thr-OH, is supplied as a white to off-white crystalline powder. The molecular formula is C₁₂H₁₅NO₅, and the formula weight is 253.25 g/mol. The product identifier distinguishes the free acid from related entries such as Cbz-Thr-OBn, Cbz-Thr(tBu)-OH, and Cbz-Thr-OMe; this monograph covers only the unprotected side-chain free acid. Standard pack sizes are 5 g, 25 g, 100 g, and 500 g in amber glass or fluoropolymer-lined closures under argon. Short-term storage at 2–8 °C in a desiccated cabinet is recommended, and the material should be equilibrated to ambient temperature under inert gas before opening to reduce surface moisture uptake. Quality documentation is issued under ISO 9001:2015 controls, with batch-specific certificate-of-analysis values for assay, polarimetry, and residue on ignition.

The unprotected secondary alcohol in the threonine side chain creates a processing boundary that is absent in glycine or alanine analogues. During carbodiimide-mediated coupling in DMF or dichloromethane, O-acylation competes with C-terminal activation when preactivation exceeds approximately 2 min at 20–25 °C. The coupling is therefore conducted at 0–4 °C with additive systems such as 1-hydroxybenzotriazole or ethyl cyano(hydroxyimino)acetate, and the activated intermediate is transferred to the free amine or resin within 90–120 seconds after addition of the carbodiimide. For manual solid-phase synthesis, pre-chilled solvent lines and jacketed reaction vessels are required; for automated synthesizers with a single-shot injection loop, the delay volume must be calibrated to avoid premature activation in the delivery line. Published data for this specific configuration is limited, but the cited window is used to reduce Cα racemization and O-acyl by-products.

How Does Cbz-Thr-OH Remain Orthogonal to Fmoc and Boc Strategies?

N-Benzyloxycarbonyl protection is removed by hydrogenolysis or by strongly acidic transfer hydrogenation, not by the base treatment used for Fmoc removal. The Cbz group therefore survives 20–25% piperidine in DMF for at least the 5–10 min periods typical of Fmoc deprotection and remains intact during TFA-mediated global side-chain deprotection at 20–25 °C for 1–2 h. Conversely, Boc protection is removed by TFA and Alloc by Pd(0)-catalyzed allyl transfer, while Cbz requires hydrogen gas or a hydrogen donor over palladium or platinum. This cleavage orthogonality allows Cbz-Thr-OH to be used as an N-terminal block in fragment condensations where the peptide backbone contains Fmoc- or Boc-protected lysine residues and the C-terminal protecting group is a benzyl ester or tert-butyl ester. The free β-hydroxy group in the standard grade is available for subsequent glycosylation or phosphorylation, provided the coupling partner is pre-formed under non-reducing conditions.

The choice of Cbz over Fmoc is generally dictated by downstream deprotection infrastructure rather than by coupling efficiency. Fmoc removal produces substantial piperidine waste, whereas hydrogenolysis generates toluene and carbon dioxide after catalyst separation. Cbz protection is selected when the peptide sequence contains base-sensitive esters or when the final protected peptide must survive acidic cleavage steps. The Cbz route is less suited to continuous-flow solid-phase synthesis because hydrogenolysis is a heterogeneous gas–liquid–solid process requiring filtration of palladium from the reactor train; Fmoc chemistry is more readily automated with liquid reagents.

Certificate-of-Analysis Parameters and Polarimetry Drift Control

Specific rotation is the most sensitive indicator of enantiomeric integrity for this material. A typical acceptance window of -4.5° ± 1.0° at 20 °C, c=1.0 in acetic acid, with sodium D-line measurement, is used. Values outside this window usually indicate residual solvent, partial deprotection, or the presence of the D-enantiomer from incomplete resolution. HPLC on a C18 stationary phase with a mobile phase consisting of 0.1% trifluoroacetic acid in water and acetonitrile at 210 nm is used for assay and impurity profiling; baseline separation of the D-enantiomer requires a chiral stationary phase or pre-column derivatization, so polarimetry is retained as a complementary lot-release test. Loss on drying is determined under vacuum at 60 °C, and residue on ignition is limited to 0.10% to control non-volatile inorganic contamination that would poison subsequent palladium catalysts.

Thermal characterization by differential scanning calorimetry shows a melting endotherm in the range 101–103 °C with decomposition above 140 °C; the melting point is not used as a primary identity criterion because traces of solvent depress the onset. Infrared spectroscopy exhibits the carbamate carbonyl stretch at approximately 1690–1710 cm^-1 and the acid carbonyl at approximately 1725–1745 cm^-1, while the broad hydroxyl absorption overlays the 2500–3300 cm^-1 region. These spectral features differentiate Cbz-Thr-OH from the corresponding methyl or benzyl ester, where the acid carbonyl is absent. For applications requiring exact stoichiometry in coupling, the free acid is dried and titrated by acid-base potentiometry using 0.1 mol/L tetrabutylammonium hydroxide in methanol.

For peptide synthesis, Cbz-Thr-OH is used primarily in solution-phase syntheses of short protected fragments. A typical workflow dissolves the protected amino acid in anhydrous dichloromethane or DMF at 0–4 °C, adds 1.0–1.1 equivalents of N,N'-diisopropylcarbodiimide or 1.0 equivalent of ethyl chloroformate in the presence of N-methylmorpholine, and then adds the amine component within 5 min. The crude protected peptide is isolated by extraction or precipitation and checked by thin-layer chromatography before hydrogenolysis. If the target peptide contains a C-terminal tert-butyl ester, the Cbz group can be removed selectively by hydrogenolysis at 0.3–0.5 MPa hydrogen without cleaving the tert-butyl ester; if the C-terminal group is a benzyl ester, catalytic hydrogenation removes both Cbz and benzyl ester, so a different orthogonal scheme is required.

When Hydrogenolysis Conditions Govern Scale-Up Feasibility

Removal of the Cbz protecting group from Cbz-Thr-OH-containing peptides is performed in a Parr-type stirred hydrogenation vessel or a continuous-flow hydrogenation cartridge with 10% palladium on carbon or palladium hydroxide on carbon. The hydrogen pressure is typically maintained at 0.3–0.5 MPa and the internal temperature at 20–25 °C. Hydrogen uptake, not elapsed time, is the endpoint criterion because catalyst activity varies between lots; fresh catalyst can complete deprotection in 30–60 min, whereas re-used catalyst may require 2–4 h or fail to reach the theoretical hydrogen volume. Catalyst poisoning by sulfur-containing impurities or heavy metals in the protected peptide must be controlled by the ≤10 ppm heavy metals specification and by confirming palladium activity on a reference substrate before charging the main batch.

The unprotected hydroxyl group of threonine is generally stable under these hydrogenation conditions; however, prolonged exposure above 25 °C or pressure above 0.7 MPa has been associated with over-reduction of the benzyl carbamate to toluene and with partial N-methylation in the presence of formaldehyde impurities from solvent degradation. This is an operational boundary rather than a routine deprotection failure. Cbz-Thr-OH should not be hydrogenated in the presence of nitro, azido, or alkenyl groups without selective poisoning agents, because the catalyst will reduce those functionalities. Transfer hydrogenation with ammonium formate or cyclohexadiene at 50–60 °C is an alternative for substrates that cannot tolerate pressurised hydrogen; the reaction is slower and requires careful control of pH to avoid formylation of the free amine.

The product is incompatible with strong aqueous alkali at elevated temperature. Storage in solution above pH 7.5 or above 25 °C accelerates carbamate hydrolysis to L-threonine and benzyl alcohol, resulting in a drop in assay and an increase in free threonine by ion-exchange chromatography. For anhydrous peptide coupling, if loss on drying exceeds 0.50%, pre-drying at 40 °C under vacuum for 16 h is required; drying above 50 °C is not recommended because the benzyl carbamate can undergo thermal decomposition at the hot vessel surface. Contact with amine-based additives during storage should be avoided, not because of premature deprotection but because moisture and residual amine accelerate carbamate solvolysis. Halogenated solvents should be removed before hydrogenolysis, as residual dichloromethane can poison palladium and increase reactor pressure.

On manufacturing lines that condense Cbz-Thr-OH with amino acid benzyl esters, batch-to-batch variance in moisture and residual solvent has required adjustment of the carbodiimide charge by 0.02–0.05 molar equivalents when the incoming loss on drying rises from 0.10% to 0.40%. This sensitivity is due to competitive hydrolysis of the activated ester. In campaigns where the Cbz-threonine intermediate was charged without pre-drying, the isolated crude peptide purity dropped by 3–6 area% on HPLC, and the main impurity was the corresponding hydrolysis acid. This process observation supports a raw-material acceptance limit of ≤0.50% loss on drying and the use of Karl Fischer titration for the reaction solvent at ≤50 ppm water before activation.