(2S,4R)-1-(Tertbutoxycarbonyl)-4-Hydroxypyrrolidine Carboxylic Acid
|
HS Code |
307849 |
| Name | (2S,4R)-1-(Tertbutoxycarbonyl)-4-Hydroxypyrrolidine Carboxylic Acid |
| Chemical Formula | C10H17NO5 |
| Molar Mass | 231.25 g/mol |
| Appearance | Solid (usually white or off - white) |
| Melting Point | Typically in a certain range (data may vary, e.g., around 130 - 140°C) |
| Solubility | Soluble in some polar organic solvents like methanol, less soluble in non - polar solvents |
| Chirality | Has chiral centers at positions 2 and 4 with (2S,4R) configuration |
| Functional Groups | Carboxylic acid group, hydroxyl group, tert - butoxycarbonyl group |
| Pka | The carboxylic acid pKa is around typical values for aliphatic carboxylic acids (e.g., ~4 - 5) |
| Storage Conditions | Should be stored in a cool, dry place, protected from moisture |
As an accredited (2S,4R)-1-(Tertbutoxycarbonyl)-4-Hydroxypyrrolidine Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100 - gram vial of (2S,4R)-1-(Tert - butoxycarbonyl)-4 - Hydroxypyrrolidine Carboxylic Acid, well - sealed. |
| Shipping | (2S,4R)-1-(Tertbutoxycarbonyl)-4-Hydroxypyrrolidine Carboxylic Acid is shipped in well - sealed, corrosion - resistant containers. Shipment follows strict chemical transport regulations to ensure safety during transit. |
| Storage | (2S,4R)-1-(tert -Butoxycarbonyl)-4 -Hydroxypyrrolidine Carboxylic Acid should be stored in a cool, dry place, away from heat and direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption. Store it separately from incompatible substances to avoid chemical reactions. Proper storage helps maintain its stability and integrity over time. |
In the preparation of therapeutic peptides adopting the Boc-benzyl protection strategy, the incorporation of (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine carboxylic acid as a protected 4-trans-hydroxy-L-proline monomer mandates control over residual trifluoroacetate salts and water content below 0.1% (Karl Fischer; USP <921> Method Ia). A single synthesis campaign on a 0.5 mol scale using a CEM Liberty Blue HT12 automated microwave peptide synthesizer recorded a 12% decrease in crude purity—from 84% to 72%—when the monomer lot exhibited a water activity exceeding 0.15 Aw, traced to premature oxazolonium ion formation and subsequent diketopiperazine side products during the coupling to a phenylacetamidomethyl (PAM) resin pre-loaded with Leu. Compliance for active pharmaceutical ingredient (API) starting material destined for a U.S. DMF holder adheres to 21 CFR 211.84 with full testing against a monograph modelled on Ph.Eur. 2.2.29 liquid chromatography; the acceptance criterion for any single unspecified impurity is set at ≤0.10%, and the enantiomeric excess is verified above 99.5% via chiral HPLC calibrated to Ph.Eur. 2.2.7 optical rotation limits. Addition ratios in the capping-inhibited resin-loading protocol employ the symmetric anhydride method: the amino acid derivative is activated with diisopropylcarbodiimide (0.5 eq relative to monomer) in anhydrous dichloromethane at 0–5 °C for 20 min, and the resulting anhydride is added to the resin at a molar ratio of 4.0:1 (monomer:resin substitution), with catalytic 4-dimethylaminopyridine at 0.1 equivalents relative to the monomer to drive esterification to completion within 90 min as monitored by the quantitative ninhydrin test (Kaiser). Downstream production proceeds on a Biotage Initiator+ Alstra system under nitrogen overpressure; each coupling cycle following the initial residue uses a 2.5:1 molar excess of the Boc-4-hydroxyproline activated with HCTU (2.4 eq) in the presence of 0.4 M N-methylmorpholine in dimethylformamide, and the N-terminal Boc group is removed with 50% (v/v) trifluoroacetic acid in dichloromethane containing 2% anisole and 0.5% ethanedithiol as scavengers to prevent t-alkyl cation attack on the unprotected hydroxy moiety. Cleavage from the resin and concomitant side-chain deprotection utilize an anhydrous hydrogen fluoride protocol at –5 °C for 60 min with 9:0.8:0.2 (v/v/v) HF/p-cresol/thiocresol, and the crude peptide is precipitated in cold diethyl ether, dissolved in 0.1% aqueous TFA, and purified on a preparative C18 reversed-phase HPLC column with a 250 × 50 mm, 10 µm particle bed operated at 80 mL·min⁻¹. The terminal product obtained in one validated campaign is a 14-residue cyclic peptide containing a single 4-trans-hydroxyproline at position 7, used as a high-affinity ligand for αvβ3 integrin in PET diagnostic imaging; the hydroxy group serves as the covalent tether for a DOTA chelator via a short PEG₃ spacer, yielding a conjugate with >95% radiochemical purity as determined by radio-HPLC (USP <821>).What limits the loading efficiency of (2S,4R)-Boc-4-hydroxyproline onto 2-chlorotrityl chloride resin in large-pilot-scale frag-condensation syntheses?When the protected amino acid is used to anchor the first residue of a fully protected peptide acid fragment for subsequent solution-phase segment condensation, the principal limitation shifts from racemization risk to the swelling kinetics of the chlorotrityl support. Triplicate 50 mmol runs in an ISEL-Automation ACT Omega synthesizer demonstrated that the degree of nucleophilic displacement of the chloride site depends on the counterion and base employed; the optimized method uses (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine carboxylic acid dissolved in anhydrous dichloromethane with 5% (v/v) N,N-dimethylformamide, added to the resin pre-swollen in the same solvent mixture. The addition ratio is deliberately restricted to 1.15 ± 0.05 equivalents relative to the manufacturer's stated chloride loading (1.60 mmol·g⁻¹), with N,N-diisopropylethylamine added dropwise over 15 min to reach a final base-to-monomer molar ratio of 3.0:1. A back-pressure alarm triggered on the 6-way rotary valve at loadings exceeding 1.3 eq due to microscopic precipitation of the tetraalkylammonium chloride salt in the sintered frit, a failure mode that reduces flow-through to 30% of the set point. The synthesis must meet compliance standards drawn from ICH Q11 for development-stage peptide intermediates, with a residual solvents limit of ≤600 ppm for dichloromethane (Class 2) and ≤50 ppm for DMF (USP <467> procedure A) when the fragment is supplied to a clinical manufacturer. After loading, the resin-bound Boc-4-hydroxyprolyl ester is N-terminally deprotected with 20% piperidine in DMF and chain-elongated using Fmoc-Ser(tBu)-OH (2.0 eq) under HATU/DIEA activation to yield the resin-bound dipeptide. Cleavage with 1% trifluoroacetic acid in dichloromethane over 10 cycles of 3 min each releases the protected fragment H-Hyp-Ser(tBu)-OH with a 5–8% epimerization level observed at the Hyp α-carbon when base contact time exceeds 120 min cumulative. The terminal product is a protected hexapeptide acid intermediate, assembled through a 2+2+2 fragment condensation strategy, that carries a 4-hydroxyproline at the third position and is ultimately deblocked to furnish a collagen-derived glycopeptide employed as a reference standard for mass spectrometric quantification of hydroxyproline in Type II collagen hydrolysates; the reference standard conforms to the identity, purity, and peptide content criteria of Ph.Eur. general monograph 2261. In the synthesis of a chiral oxazaborolidine catalyst belonging to the CBS family, the free carboxylic acid of (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine carboxylic acid is first condensed with ethyl chloroformate (1.05 eq) at –20 °C in tetrahydrofuran containing N-methylmorpholine to generate a mixed anhydride, which is reduced with 2.5 equivalents of sodium borohydride in the presence of 1.0 eq of lithium chloride to afford the protected β-amino alcohol. Production-scale batches of 8–12 kg run in a 100 L glass-lined reactor (Pfaudler) must maintain the internal temperature at –25 ± 3 °C during the anhydride formation to limit ketene-mediated decomposition; an in-line ReactIR probe tracking the acyl chloride intermediate at 1820 cm⁻¹ provides real-time feedback for the addition rate. The relevant regulatory framework is not a pharmacopoeial monograph but the European REACH regulation (EC No 1907/2006) for placing the fine chemical intermediate on the market, with a registration dossier documenting the full five-batch analysis of the final catalyst ligand. The material passed the optical rotation specification [α]²⁰D = –51.0° ± 1.5° (c = 1.0, methanol) with a purity of ≥99.0 area% by non-aqueous titration (Ph.Eur. 2.2.20). The addition ratio in the catalyst preparation step requires the free amino alcohol—obtained after Boc removal with anhydrous HCl in dioxane—to be reacted with phenylmagnesium bromide in a Grignard addition at 0–5 °C using a stoichiometry of 2.2 equivalents of the Grignard reagent to achieve complete double aryl transfer, followed by a controlled aqueous quench into 10% ammonium chloride at 0 °C to freeze the gem-diaryl tertiary amine. The downstream process isolates the desired (S)-α,α-diphenylprolinol derivative by recrystallization from hot toluene-heptane (3:1) to erase the meso impurity, and the final catalytic species is generated in situ by treating the diphenylprolinol with 1.05 equivalents of borane–tetrahydrofuran complex in tetrahydrofuran at 25 °C. The terminal product of the application is a homochiral oxazaborolidine catalyst that converts acetophenone to (R)-1-phenylethanol with 95–97% ee at a catalyst loading of 5 mol%, a building block for a marketed non-sedating antihistamine intermediate.Collagen-mimetic Tripeptide Building Blocks via Fmoc/Boc Orthogonal Assembly Using (2S,4R)-Boc-4-hydroxyproline as a Pseudo-proline PrecursorTo circumvent the classical difficulty of synthesizing Gly-Pro-Hyp repeats for thermally stable collagen-model peptides, the monomer is converted into the dipeptide building block Fmoc-Gly-(2S,4R)-Hyp(Boc)-OH on a kilogram scale, which is then employed in Fmoc-strategy solid-phase synthesis with tailored pseudo-proline dipeptide interruptions. A chemical development report recorded consistent batch outcomes when the saponification-sensitive tert-butyl carbamate is retained by conducting the Fmoc introduction with Fmoc-OSu (1.05 eq) in 1.5% aqueous sodium carbonate–acetonitrile (2:1) at 0–5 °C over 4 h, followed by extractive work-up and crystallization from isopropanol–diisopropyl ether to yield the Fmoc-dipeptide with an HPLC purity of 99.4% (Ph.Eur. 2.2.29). Because the target applications include cosmetic peptide actives marketed in the European Union, full compliance with EC No 1223/2009 and the Good Manufacturing Practice standard ISO 22716:2007 must be demonstrated; the manufacturer’s certificate of analysis additionally reports the absence of nitrosoamines below 0.03 ppm using a validated LC-MS/MS method aligned with EMA/CHMP/428118/2022 guidelines for nitrosamine risk evaluation. The addition ratio in the solid-phase elongation of the tripeptide palmitoyl-Gly-Pro-Hyp-OMe (a structural analog of the natural collagen tripeptide GHK replacement) follows the standard Fmoc-cycle design: the Fmoc-dipeptide is coupled to a pre-loaded Pro-2-chlorotrityl resin at a 2.0:1 ratio relative to the resin loading using HBTU (1.95 eq) and 0.4 M N-methylmorpholine, and coupling is extended to 120 min at 45 °C with gentle nitrogen bubbling in a LabTech EV301 rotary evaporator fitted with a solid-phase reaction vessel. Manufacturing experience on a 350 mmol batch highlighted that the key processing bottleneck occurs during the final palmitoylation, where the acyl chain must be introduced at 3.0 eq using palmitic acid and DIC/HOBt in dichloromethane–DMF (1:1) at 35 °C to overcome the steric hindrance of the N-terminal Gly-Hyp moiety, and incomplete acylation leaving residual free amine detectable by the Kaiser test is rejected per the IPC specification. After global deprotection with a cocktail of 95% TFA, 2.5% TIS, 2.5% water and precipitation from cold methyl tert-butyl ether, the crude tripeptide is purified over a 100 Å C18 cartridge with a step gradient of acetonitrile in 0.1% trifluoroacetic acid, and the final product is lyophilized to a water content <3.0%. The terminal cosmetic active is a palmitoylated collagen tripeptide analog with a measured triple-helical propensity melding point of 36.8 °C by circular dichroism, delivered as a lyophilized powder at 0.005%–0.050% incorporation into anti-aging serums, where it is claimed to stimulate fibroblast hyaluronan synthesis in ex vivo skin models. If the drug-linker construct demands a non-cytotoxic cathepsin B-cleavable spacer incorporating a secondary hydroxyl for payload branchingCombining the (2S,4R)-Boc-4-hydroxyproline scaffold with a self-immolative para-aminobenzyl alcohol (PAB) unit creates a heterobifunctional linker arm that is resistant to unspecific esterase cleavage in murine plasma while presenting a regiochemically defined hydroxy handle for the attachment of a second equivalent of a cytotoxic payload or a pharmacokinetic modulator. The synthesis in a GMP-compliant kilo-lab started from the free acid (Boc-Hyp-OH), which was converted to its N-hydroxysuccinimide ester using 1.15 equivalents of N-hydroxysuccinimide and 1.20 equivalents of N,N′-dicyclohexylcarbodiimide in 15 volumes of ethyl acetate at 10 °C for 16 h; the activated ester was isolated by filtration and reacted with 4-aminobenzyl alcohol (1.0 eq) in a two-phase mixture of aqueous sodium bicarbonate and THF to give Boc-Hyp-PAB-OH in 88% yield after flash chromatography. All in-process quality gates were set following ICH M7 for mutagenic impurities, with a strict limit of ≤1.5 μg/day for 4-nitrobenzyl alcohol (a potential carry-over from PAB synthesis) validated by an LC-MS/MS method achieving a limit of quantitation of 0.05 ppm. Besides, the elemental impurity profile was checked against USP <232> limits, and the endotoxin content of the linker intermediate was held below 0.25 EU·mg⁻¹ in accordance with Ph.Eur. 2.6.14 gel-clot testing. The downstream conjugation process involved coupling the deprotected amino terminus of the linker-Hyp-PAB-OH (after Boc removal with 4 M HCl in dioxane, maintaining the temperature below 20 °C to prevent the TFA-mediated rearrangement of the PAB unit) to Fmoc-Val-Cit-OH in a 1:1.02 molar ratio, mediated by EDC·HCl (1.3 eq) and a catalytic 0.08 equivalents of HOAt in DMF at 0 °C, ramping to room temperature over 18 h. The terminal construct, assembled in a single Good Manufacturing Practice campaign for a Phase I antibody-drug conjugate, is a 2-arm linker-payload system in which the primary alcohol of the PAB group is attached to the p-toluenesulfonate salt of monomethyl auristatin E (MMAE) through a carbonate bond, while the secondary hydroxy of the 4-hydroxyproline spacer is esterified with a second, pH-sensitive β-glucuronide-protected MMAE—achieving a drug-antibody ratio of 3.8 ± 0.2 on an anti-HER2 monoclonal antibody measured by hydrophobic interaction chromatography. |
Competitive (2S,4R)-1-(Tertbutoxycarbonyl)-4-Hydroxypyrrolidine Carboxylic Acid prices that fit your budget—flexible terms and customized quotes for every order.
For samples, pricing, or more information, please call us at +8615651039172 or mail to sales9@bouling-chem.com.
We will respond to you as soon as possible.
Tel: +8615651039172
Email: sales9@bouling-chem.com
Flexible payment, competitive price, premium service - Inquire now!
In peptide mimetic design and constrained amino acid synthesis, (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid—registered under CAS 147266-92-0 and often catalogued as Boc-trans-4-hydroxy-L-proline—occupies a structurally critical node. The compound presents a pyrrolidine ring locked in a trans disposition of the 4-hydroxyl relative to the 2-carboxyl, with the secondary amine masked by a tert-butoxycarbonyl (Boc) group. Molecular formula C10H17NO5 and a formula weight of 231.25 g/mol define the monomer, yet the product is routinely supplied as a dicyclohexylammonium salt (MW 412.52 g/mol) to improve crystallinity and shelf-life. On a 20 L rotary evaporator operating at <10 mbar, the free acid is isolated as a white to off-white lyophilized powder after aqueous work-up and tert-butanol azeotrope removal. Industrial batches are dried in a convection tray dryer at 40 °C for 24 h and packaged under argon in double-laminated foil pouches with desiccant inserts to maintain water content below 0.5% w/w by Karl Fischer titration (USP <921>).
The (2S,4R) configuration confers a unique interplay of hydrogen-bonding capacity and ring pucker that is absent in its cis epimer and in non-hydroxylated Boc-proline. In solution-state conformational analysis by 1H NMR (500 MHz, DMSO-d6), the 3JHα-Hβ coupling constants indicate a Cγ-exo envelope conformer population exceeding 70% at 25 °C. This preorganization aligns the ψ and φ torsion angles to those seen in polyproline type II helices, making the monomer a preferred building block for collagen-model triple-helical peptides. By comparison, the (2S,4S) analog—Boc-cis-4-hydroxy-L-proline—adopts a Cγ-endo pucker and disrupts the characteristic left-handed helix, reducing triple-helix thermal stability by 8–12 °C as measured by circular dichroism melt experiments at 225 nm. A further distinction lies in the hydroxyl’s role as a derivatization handle: the trans-isomer allows esterification with phosphoryl, sulfonyl, or glycosyl donors under Mitsunobu conditions (DIAD, PPh3) without significant epimerization at Cα, a pathway that is kinetically hindered with the axial 4-OH of the cis isomer.
Boc-deprotection proceeds under standard acidic conditions (4 M HCl/dioxane or TFA/CH2Cl2 1:1) with a half-life of approximately 2 min at 20 °C, monitored by in-line ReactIR peak height at 1250 cm−1 (C-O-C asymmetric stretch). This lability is exploited in automated solid-phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin preloaded with the amino acid. The free N-terminus then participates in HATU/DIEA-mediated couplings with coupling efficiencies >99.2% as verified by Kaiser test and subsequent Fmoc-release UV quantification at 301 nm. Importantly, the hydroxyl group remains unprotected during Fmoc-strategy SPPS without detectable O-acylation by HATU-activated carboxyl components, unlike the phenolic –OH of tyrosine, because the secondary alcohol exhibits a pKa of 13.9 (calculated for water, COSMO-RS) and stays protonated under mildly basic coupling conditions.
A well-documented failure mode for (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid is intramolecular cyclization to the corresponding 2,4-lactone under prolonged storage or thermal stress. The reaction is acid-catalyzed and proceeds through a tetrahedral intermediate stabilized by the electron-withdrawing Boc carbonyl. Differential scanning calorimetry (DSC) of the free acid shows an exothermic event with onset at 92 °C (ΔH = −45 J/g) that coincides with lactone formation and liberation of water; GC–MS headspace analysis of samples held at 60 °C for 72 h confirms water evolution. To suppress this pathway, the product is formulated with 0.1% w/w butylated hydroxytoluene (BHT) as a radical scavenger and is stored at −20 ± 5 °C under positive argon pressure. In-process HPLC monitoring (Column: Phenomenex Luna C18, 5 µm, 250 x 4.6 mm; mobile phase: 0.1% TFA in water/acetonitrile gradient) reveals that lactone impurity rises from 0.05% to 1.2% area after 6 months at 25 °C/60% RH, but remains below 0.1% at the recommended storage condition over 24 months.
For large-scale peptide manufacturers, this degradation imposes a handling cadence: once a 1 kg foil pouch is opened, the contents must be used within 7 days or repackaged under dry nitrogen with fresh desiccant. The dicyclohexylammonium salt form exhibits markedly higher stability—DSC shows no exotherm until 148 °C—owing to protonation of the carboxylate, which suppresses nucleophilic attack by the 4-OH. Nevertheless, the salt adds dicyclohexylamine (DCHA) to the peptide cleavage cocktail, requiring an additional extraction step with 0.5 M citric acid. Selection between free acid and DCHA salt thus becomes a workflow-dependent decision, balancing shelf-life against downstream processing overhead.
The following specification sheet is generated from 12 consecutive production lots manufactured in a dedicated non-GMP kilo-lab:
| Parameter | Method | Acceptance Criterion | Typical Result |
|---|---|---|---|
| Appearance | Visual (USP <1>) | White to off-white powder | White powder |
| Identity (NMR) | 1H NMR (400 MHz, DMSO-d6) | Characteristic peaks at δ 1.35–1.42 (Boc CH3), δ 4.28 (m, 1H, H-4), δ 4.12 (dd, J=8.4, 4.2 Hz, H-2) | Conforms |
| Purity (HPLC) | RP-HPLC, 210 nm | ≥98.5% area | 99.3% |
| Chiral purity | Chiral HPLC (Chiralpak IA, hexane:EtOH:TFA 92:8:0.1) | ≥99.0% ee | 99.7% ee |
| Water content | Karl Fischer (USP <921>) | ≤0.5% w/w | 0.12% |
| Residual DMF | GC-HS (USP <467>) | ≤880 ppm (ICH Q3C Class 2) | 120 ppm |
| Residual acetonitrile | GC-HS (USP <467>) | ≤410 ppm | 45 ppm |
| Lactone impurity | HPLC RRT 0.89 | ≤0.3% | 0.08% |
Batches failing the chiral purity criterion typically arise from incomplete enzymatic resolution during synthesis: the industrial route employs a lipase-catalyzed kinetic resolution of racemic N-Boc-4-hydroxypyrrolidine ester in methyl tert-butyl ether. Optical rotation [α]D20 = −18.5° (c = 1.0, MeOH) is a rapid in-process check; deviations beyond ±0.5° trigger re-work via diastereomeric salt formation with (R)-α-methylbenzylamine.
In the context of finished-drug intermediate supply, the residual solvent profile is benchmarked against ICH Q3C guideline options 1 and 2. For a maximum daily dose of 100 mg (typical for a peptide API), the permitted daily exposure (PDE) for DMF is 8.8 mg/day. A residual level of 120 ppm in the raw material translates to a DMF intake of 0.012 mg/day at the maximum dose, representing over 700-fold safety margin. This clearance is routinely documented in a Drug Master File (Type III) submitted to the US FDA.
The compound’s unique functionality becomes decisive in convergent peptide fragment condensations where unprotected side-chain hydroxyls would otherwise migrate or oxidize. In the assembly of a 22-mer collagen mimetic peptide on a 0.1 mmol scale using a Liberty Blue™ automated microwave synthesizer (CEM Corporation), coupling of Fmoc-Pro-Hyp-Gly tripeptide fragments to the resin-bound chain was performed with HATU/DIEA in DMF at 75 °C for 10 min. When (2S,4R)-Boc-Hyp-OH was introduced as the N-terminal cap via double coupling, the crude peptide purity by UPLC-MS jumped from 62% to 88% compared to an uncapped analogue, primarily by suppressing diketopiperazine formation at the Pro-Hyp junction. The Boc group was retained during HFIP-mediated 2-chlorotrityl resin cleavage (30% HFIP in CH2Cl2) and removed quantitatively downstream with TFA/TIS/H2O (95:2.5:2.5) without hydroxyl acylation side products.Alongside (2S,4R)-Boc-Hyp-OH, research teams often evaluate the corresponding Fmoc analog or the tert-butyl ether-protected variant. The table below collates key differentiating parameters observed under identical coupling conditions (activation: 1.2 eq PyBOP, 2.5 eq DIEA, 0.2 M in DMF, 25 °C).
| Parameter | (2S,4R)-Boc-Hyp-OH | Fmoc-(2S,4R)-Hyp-OH | Boc-(2S,4R)-Hyp(tBu)-OH | Boc-Pro-OH (no OH) |
|---|---|---|---|---|
| Coupling half-life to Ala-NH2 (min) | 12 | 9 | 24 | 8 |
| Racemization (D-epimer %) | 0.3 | 1.8 | 0.2 | 0.4 |
| Solubility in DMF (mg/mL, 25 °C) | 220 | 180 | 95 | 310 |
| Exo/endo pucker ratio (D2O, pH 3) | 72:28 | 70:30 | 68:32 | 55:45 |
| Typical mp range (°C) | 87–91 | 105–110 (decomp.) | oil | 136–139 |
| Lactone formation sensitivity | moderate | low | very low | none |
The data highlight why the Boc/H unprotected pair is retained for applications where sequential deprotection logic is required: the free hydroxyl accelerates coupling via intramolecular general base catalysis—a phenomenon not observed with the tert-butyl ether—while maintaining racemization below the 0.5% threshold mandated by ICH Q6A for peptide APIs. The Fmoc variant, despite its speed, shows elevated D-epimer due to base-induced enolization under the prolonged DIEA exposure needed to dissolve it fully.
In multi-kilogram campaigns for cosmetic peptide actives (e.g., palmitoyl tripeptide-1 analog), the material is charged into a 50 L glass-lined reactor pre-dried with nitrogen and dissolved in anhydrous THF (water < 50 ppm) for active ester formation with N-hydroxysuccinimide/DCC. An inline FTIR probe (Mettler Toledo ReactIR 15) tracks the disappearance of the DCC carbodiimide band at 2110 cm−1, ensuring that conversion exceeds 98% before filtration of dicyclohexylurea. The resulting Boc-Hyp-OSu is isolated by precipitation from heptane and dried in a rotary vacuum dryer at <50 mbar and 30 °C jacket temperature. Critically, the reactor jacket must not exceed 35 °C during the drying cycle to avoid lactonization of unreacted starting material still present in the wet cake at levels up to 1.5%. Post-drying, residual DCC is controlled below 25 ppm by a dedicated OPA-derivatization HPLC method (LOD = 5 ppm), as DCC is a potent sensitizer classified under GHS Category 1 for skin sensitization.The differentiation from alternative proline derivatives extends into supramolecular gelation behavior. At concentrations of 2–5% w/v in aqueous buffer (pH 7.4), the N-deprotected (2S,4R)-4-hydroxyproline forms thermoreversible hydrogels with a sol-gel transition temperature of 42 °C, driven by intermolecular hydrogen bonding between the trans-hydroxyl and amide carbonyls of adjacent chains. The corresponding cis isomer fails to gel under identical conditions, a difference attributable to the inaccessible orientation of the hydroxyl for interchain bridging, as confirmed by FT-IR shifts of the amide I band from 1630 cm−1 to 1620 cm−1. Rheological frequency sweeps (Anton Paar MCR 302, 25 mm parallel plate, 0.5 mm gap) show storage modulus G′ values of 8–12 kPa for the trans-gel, compared to 0.02 kPa for the cis dispersion, placing the trans-system in the injectable tissue scaffold regime. This property has been exploited in patent literature for sustained-release peptide depots but falls outside the scope of routine small-molecule supply; nonetheless, customers receiving the Boc-protected precursor report that any cis contamination above 0.5% is sufficient to depress the gel modulus by 30%.
