| HS Code | 419706 |
| Product Name | BOC-L-Proline |
| Synonyms | N-(tert-Butoxycarbonyl)-L-proline; (S)-1-(tert-Butoxycarbonyl)pyrrolidine-2-carboxylic acid |
| Cas Number | 15761-39-4 |
| Molecular Formula | C10H17NO4 |
| Molecular Weight | 215.25 g/mol |
| Appearance | White to off-white crystalline powder |
| Melting Point | 132-135 °C |
| Optical Rotation | [α]20/D = -60.0° (c=1, methanol) |
| Solubility | Soluble in methanol, ethanol, dimethylformamide, dichloromethane; sparingly soluble in water |
| Storage Conditions | Store in a cool, dry place, preferably at 2-8 °C, protected from moisture |
| Purity | ≥99% (TLC) |
| Mdl Number | MFCD00037312 |
| Smiles | CC(C)(C)OC(=O)N1CCCC1C(=O)O |
As an accredited BOC-L-Proline factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | BOC-L-Proline, 5 g, supplied in a sealed amber glass bottle with tamper-evident cap, stored under cool, dry conditions. |
| Container Loading (20′ FCL) | 20′ FCL: packed in drums/cartons on pallets, secured with straps, moisture-protected, labeled, container sealed for safe transit. |
| Shipping | BOC-L-Proline (N-Boc-L-proline) is shipped as a stable crystalline solid at ambient temperature in sealed, moisture-resistant containers. Avoid excessive heat and humidity during transit. Ensure compliance with local regulations; handle with standard laboratory precautions. For research use only, not for human consumption. |
| Storage | Store BOC-L-Proline in a tightly sealed container under refrigerated conditions (2–8°C), protected from moisture and direct light. Keep the container desiccated and allow it to reach room temperature before opening to prevent condensation. Avoid prolonged exposure to air, as hygroscopic absorption may reduce purity and stability over time. |
| Shelf Life | Store at 2–8°C, tightly sealed, desiccated, away from light. Shelf life is typically two years from manufacture if unopened. |
The use of BOC-L-Proline (CAS 15761-39-4; Mw 215.25 g/mol) in Boc-SPPS manufacturing campaigns for therapeutic peptide APIs is as a protected imino acid building block, not a formulation additive, and its addition ratio is therefore expressed as molar equivalents relative to resin-bound amine. Standard production coupling loads BOC-L-Proline at 3–5 mol per mol of aminomethyl polystyrene or 4-methylbenzhydrylamine resin substitution, with the resin substitution typically 0.6–1.2 mmol/g. The acid is dissolved in anhydrous DMF or N-methyl-2-pyrrolidone at 0.25–0.5 M, preactivated with HATU at 0.95–1.0 mol per mol acid and DIPEA at 2.0–2.5 mol per mol acid, and circulated through the SPPS reactor for 45–60 min at 20–30 °C. Process validation follows ICH Q7 Section 12.4, and incoming raw material release includes identity by USP <781> optical rotation, purity by USP <621> HPLC, and residual solvent compliance with USP <467> and ICH Q3C. The downstream production process uses jacketed SPPS vessels from 50 L to 1,500 L with nitrogen blanketing and variable-speed agitation; resin swelling in DCM reaches 3–5×, so the vessel freeboard must remain above 30%. A process conflict occurs after TFA-mediated Boc removal when the newly exposed N-terminal proline secondary amine is acylated by the next amino acid: the reduced nucleophilicity of the cyclic amine leads to slower coupling, incomplete sequences, and deletion impurities that cannot be removed by simple recrystallization. In those positions, the next amino acid is double-coupled at 0.5 M in NMP with PyBOP or HATU at 40 °C for 60 min, and residual free amine is capped with acetic anhydride/pyridine (1:1) for 10 min. In-process monitoring by Kaiser test is less reliable at proline residues because the imino acid gives a weak color response; consequently, pilot-scale campaigns add HPLC-MS analysis after the proline coupling and after the following coupling. Finished products are proline-bearing therapeutic peptide APIs, usually isolated as lyophilized acetate or hydrochloride salts, with unspecified impurity thresholds controlled under ICH Q3A(R2). Published data for specific peptide sequences remains limited due to drug master file confidentiality, but the coupling behavior of BOC-L-Proline on the production-scale equipment described above is consistent across generic peptide CMO technical packages.
| Resin-bound amine scale | BOC-L-Proline equivalents | Activation system | Concentration in DMF | Reaction window |
|---|---|---|---|---|
| 0.1 mmol | 4.0 | HATU/DIPEA | 0.25 M | 20–25 °C, 45 min |
| 500 mmol | 3.5 | HATU/DIPEA | 0.40 M | 25–30 °C, 60 min |
| 5 mol | 3.0–4.0 | HBTU/HOBt/DIPEA | 0.30 M | 20–25 °C, 60 min, double coupling if Kaiser positive |
For solution-phase assembly of proline-containing cardiovascular peptidomimetic APIs, BOC-L-Proline is retained as the N-protected acid until the final prolyl amide bond is formed, because premature Boc removal releases the free proline zwitterion that is poorly soluble in anhydrous tetrahydrofuran and causes emulsion formation during aqueous workup. The addition ratio in mixed anhydride activation is 1.05–1.2 mol of BOC-L-Proline per mol of amine intermediate, with pivaloyl chloride at 1.05–1.1 mol per mol of acid and N-methylmorpholine at 1.2–1.4 mol per mol of acid, at a jacket temperature of −15 °C to −10 °C. The downstream production process is carried out in a glass-lined reactor equipped with a bottom drain and pH-controlled quench; after coupling, the reaction mass is quenched with 5% aqueous citric acid, extracted into ethyl acetate, washed with 5% sodium bicarbonate, and solvent-swapped to isopropanol for crystallization. Residual tetrahydrofuran and ethyl acetate are controlled under ICH Q3C and USP <467>; unspecified impurities in the isolated proline-containing intermediate are held at ≤0.10% under ICH Q3A(R2). The terminal finished product types are proline-bearing peptidomimetic API intermediates or crude APIs, subsequently converted to crystalline salts for cardiovascular drug product formulation. A critical boundary is the coupling temperature: below −15 °C the mixed anhydride formation becomes incomplete, while above −5 °C disproportionation of the mixed anhydride and racemization side reactions increase, though published kinetic data for specific cardiovascular intermediates under exact production-scale conditions is limited.
At the 100 L pilot scale, BOC-L-Proline is converted into chiral diarylprolinol ligands through a sequence of esterification, N-Boc protection maintenance, and aryl Grignard addition. The stoichiometric addition ratio for the Grignard step is 2.2–3.0 mol of aryl magnesium halide per mol of BOC-L-proline methyl ester, dosed at −10 °C to 0 °C over 4–6 h; esterification of the carboxylic acid is conducted with thionyl chloride in methanol at 0–5 °C using 1.2–1.5 mol thionyl chloride per mol of BOC-L-Proline. The downstream production process requires a nitrogen-purged, jacketed glass-lined vessel with a controlled dosing pump and in-process FTIR for carbonyl disappearance; the aryl Grignard addition is exothermic and the reactor jacket must maintain a thermal gradient below 5 °C per minute during dosing. After quench with saturated ammonium chloride and extraction into methyl tert-butyl ether, the product is crystallized from heptane/ethyl acetate. Compliance for non-pharmaceutical organocatalyst manufacture is governed by ISO 9001:2015 and REACH registration dossiers; residual solvents are tested by USP <467> where the product is supplied to pharmaceutical laboratories. The terminal finished product types are chiral diarylprolinol silyl ethers and related L-proline-derived organocatalysts used for asymmetric Diels–Alder, aldol, and epoxidation reactions. The main process limitation is temperature control during Grignard addition; above 0 °C the selectivity erodes, but published enantiomeric excess data for this specific protected proline substrate under exact production-scale conditions is limited.
Cosmetic peptide synthesis contractors running solution-phase fragment lines use BOC-L-Proline at 1.05–1.2 mol per mol of amine fragment in anhydrous ethyl acetate or THF with pivaloyl chloride mixed anhydride activation at −10 °C to 0 °C, followed by aqueous quench, phase separation, and preparative HPLC purification to ≥95% purity; the proline-containing oligopeptide concentrates are released under ISO 22716:2007 cosmetic GMP and evaluated for residual solvents according to USP <467>, while the final topical formulations containing these peptide active ingredients are regulated under EC 1223/2009. Finished products are lyophilized proline-containing oligopeptide concentrates supplied to topical formulators, where the peptide is incorporated into serums and creams at 0.001–0.01% w/w of the dry peptide content in the final cosmetic formulation.
Because research-grade synthesis laboratories operate under different release criteria than GMP API facilities, BOC-L-Proline is used in solution-phase parallel synthesis of proline-containing peptide libraries where orthogonal protection and acid-labile intermediate handling are required. The addition ratio is 1.2–1.5 mol of BOC-L-Proline per mol of amine-functionalized synthon, using propylphosphonic anhydride at 1.5 mol per mol of acid and diisopropylethylamine at 3.0 mol per mol of acid in ethyl acetate at 20–25 °C; coupling progression is monitored by UPLC-MS after 60 min and again after 120 min. The downstream production process is executed on automated liquid handlers or bench-scale jacketed vessels with disposable static mixers, and each well or batch is tracked by ISO 9001:2015 documentation with high-resolution LC-MS and NMR release data. Residual BOC-L-Proline and coupling reagents are removed by solid-phase extraction or preparative HPLC, using a binary acetonitrile/water gradient with 0.1% trifluoroacetic acid. The terminal finished product types are lyophilized peptide library arrays and custom research reagents supplied to academic screening centers and pharmaceutical discovery units, with compound identity confirmed by HRMS and purity specified at ≥90% for primary screening.
The conversion of BOC-L-Proline to its N-hydroxysuccinimide ester introduces a moisture-sensitive functional group that places strict handling boundaries on downstream bioconjugation and affinity-resin manufacturing. The addition ratio is 1.05–1.2 mol N-hydroxysuccinimide and 1.05–1.1 mol dicyclohexylcarbodiimide per mol of BOC-L-Proline, in anhydrous ethyl acetate or dichloromethane at 0–5 °C for 4–8 h; the resulting N,N'-dicyclohexylurea is removed by filtration through a 0.2 µm filter. The downstream production process uses a nitrogen-purged reactor and moisture traps because the preactivated ester hydrolyzes to BOC-L-Proline when exposed to ambient relative humidity above 40%, releasing N-hydroxysuccinimide and reducing coupling efficiency in subsequent peptide conjugation. Compliance for these preactivated esters is governed by ISO 9001:2015 and, when supplied for pharmaceutical conjugation, residual solvent and impurity testing under USP <467> and ICH Q3C; the finished product type is a lyophilized or crystalline activated ester used to conjugate proline residues to amino-functionalized peptides, proteins, or chromatography resins. Process operators control packaging under nitrogen with desiccant and specify storage below −20 °C in sealed amber glass, because repeated freeze-thaw cycles increase free acid content, although published data for this specific configuration is limited.
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BOC-L-Proline, also specified as N-(tert-butoxycarbonyl)-L-proline or Boc-Pro-OH, is supplied as a white to off-white crystalline powder with molecular formula C10H17NO4, CAS 15761-39-4, and molecular weight 215.25 g/mol. The product is released against a specification that includes assay ≥99.0% by HPLC, enantiomeric excess ≥99.0% by chiral HPLC, specific optical rotation [α]D20 = −60° ± 2° (c = 1, acetic acid), and melting range 130–136 °C. The pyrrolidine nitrogen is masked by the acid-labile tert-butoxycarbonyl group, while the carboxyl group remains free for amide bond formation. This protection pattern distinguishes BOC-L-Proline from L-proline, Cbz-L-proline, and Fmoc-L-proline in deprotection chemistry, solubility, and solid-phase peptide synthesis compatibility. Standard pack sizes are 25 g, 100 g, 500 g, 1 kg, and 5 kg in nitrogen-purged HDPE bottles, with research grade, peptide-synthesis grade, and low-endotoxin grade available under the same core identity but different residual solvent and elemental impurity documentation.
| Parameter | Typical specification | Analytical method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection |
| Assay | ≥99.0% | HPLC, C18, 214 nm |
| Enantiomeric excess | ≥99.0% | Chiral HPLC, amylose-based column |
| Melting range | 130–136 °C | Capillary or DSC |
| Specific optical rotation | −60° ± 2° (c = 1, acetic acid) | Polarimetry, 589 nm |
| Loss on drying | ≤0.5% | Vacuum oven, 70 °C, 4 h |
| Residual solvents | Class 3 limits | USP <467> headspace GC-FID |
| Elemental impurities | ≤20 ppm | USP <233> ICP-MS |
Routine lot-release data are generated on C18 reversed-phase HPLC columns with UV detection at 214 nm and a mobile phase of 0.1% trifluoroacetic acid in water/acetonitrile. Chiral purity is controlled on an amylose-based chiral stationary phase with hexane/ethanol modifiers; residual solvents are measured by headspace GC-FID according to USP <467>, and elemental impurities are measured by ICP-MS according to USP <233>. The certificate of analysis reports actual values for assay, optical rotation, water content, and residual solvent rather than pass/fail statements alone.
Residual water exerts the primary degradation pressure on BOC-L-Proline. The compound remains stable as a dry crystalline powder under nitrogen, but exposure to relative humidity above 60% at 20 °C can produce detectable free L-proline through slow acidolysis of the Boc group. Accelerated stability studies following ICH Q1A at 40 °C/75% RH demonstrate assay loss, but published data for this specific configuration is limited and the observed magnitude is lot-dependent. Bulk handling therefore uses nitrogen-purged double polyethylene liners inside fiber drums, and process areas maintain dew point below −30 °C. The material should not be micronized in air-jet mills without dew-point control; mechanical milling can generate amorphous surfaces that adsorb water and increase residual water above the 0.5% loss-on-drying limit. In downstream reaction vessels, residual water in DMF should be controlled below 100 ppm by activated molecular sieves before adding coupling reagents, because carbodiimide activation in the presence of water can convert the carboxylic acid to an unreactive N-acylurea and release L-proline by-product. Avoid combining bulk BOC-L-Proline with strong bases or amines during storage; salt formation is exothermic and may produce localized decomposition.
In solid-phase peptide synthesis, BOC-L-Proline is used in the Boc/benzyl protection strategy, where the N-terminal Boc group is removed with trifluoroacetic acid and side-chain protection survives until final cleavage with hydrogen fluoride or trifluoromethanesulfonic acid. The proline residue is coupled on polystyrene resin crosslinked with 1–2% divinylbenzene, with resin loading between 0.3 mmol/g and 1.0 mmol/g. Couplings are performed in a manual peptide synthesis vessel or automated synthesizer with 4 equivalents of BOC-L-Proline, 4 equivalents of HBTU, and 8 equivalents of DIPEA in DMF at 20 °C. The acid-labile Boc group survives repeated neutralization and DMF washing, but prolonged exposure to residual TFA can prematurely deprotect the next coupling site; therefore each deprotection is limited to 2 × 5 min using 50% TFA in dichloromethane. Fmoc-L-proline is not compatible with this regime because the base-labile Fmoc group would be removed by piperidine or other secondary amines used during neutralization. BOC-L-Proline therefore permits proline introduction into sequences that already contain base-sensitive functionality or Fmoc-protected lysine residues when selective deprotection is required.
Solution-phase amide coupling with BOC-L-Proline is conducted by dissolving the acid in dichloromethane or DMF and activating with EDC·HCl or DIC in the presence of HOBt or Oxyma. A typical laboratory-scale reaction charges 1.0 equivalent of amine, 1.05 equivalents of BOC-L-Proline, 1.2 equivalents of EDC·HCl, 1.2 equivalents of HOBt, and 3.0 equivalents of N-methylmorpholine at 0–5 °C; the mixture is allowed to warm to 20 °C over 16 h. Conversion is monitored by TLC using ethyl acetate/hexane 1:1 and by HPLC. The proline ring imposes a turn conformation that modifies the retention time and crystallinity of downstream amide intermediates. In a 20 L jacketed reactor with retreat-curve impeller agitation at 150 rpm, addition of EDC·HCl as a solid in 5 portions over 30 min limits the temperature rise to 2 °C above jacket set point and prevents formation of the rearranged N-acylurea impurity. The resulting BOC-protected peptide ester is extracted with ethyl acetate, washed with 1 M citric acid and 5% sodium bicarbonate, and concentrated below 35 °C. This sequence is compatible with multi-kilogram batches because BOC-L-Proline remains a free-flowing crystalline solid that does not require distillation or chromatographic purification.
BOC-L-Proline becomes the preferred N-protected proline source when the target intermediate contains hydrogenation-sensitive groups or Cbz-protected amines that must be retained. Unlike Cbz-L-proline, which requires catalytic hydrogenation or strong acid and can reduce pendant benzyl ethers, BOC-L-Proline is cleaved with anhydrous acid under nonhydrogenolytic conditions. In a convergent synthesis of a constrained proline-containing peptide isostere, the Boc group is removed with 4 M HCl in dioxane at 10 °C over 30 min, while a benzyl ester on the C-terminus remains intact. The reversed-phase HPLC trace of the crude deprotected product shows less than 0.5% diketopiperazine formation when neutralization is performed with cold sodium carbonate. By comparison, Fmoc-L-proline would require piperidine, which can hydrolyze methyl esters, and Cbz-L-proline would require hydrogen over palladium on carbon, which can reduce carbon-carbon double bonds elsewhere. This orthogonality is exploited in process routes where a benzyl ester or Cbz-protected amine must remain untouched until a later stage, making BOC-L-Proline the only proline derivative among the common protected forms that is removed under strictly acidic conditions while preserving hydrogenolysis-labile groups.
Relative to Fmoc-L-proline, BOC-L-Proline has a lower molecular weight (215.25 g/mol versus 337.37 g/mol), a less bulky N-terminal protecting group, and greater solubility in ethyl acetate. The lower mass reduces the weight of protected amino acid required per mole of resin sites, although coupling efficiency in Boc-SPPS depends on TFA removal rather than piperidine deprotection. Cbz-L-proline is often supplied as a low-melting solid or syrup and is removed by hydrogenolysis, making it less convenient for acid-labile substrates. Boc-D-proline is the enantiomer with specific optical rotation approximately +60° (c = 1, acetic acid) and is selected only when the D configuration is required for biological target binding; mixing the two enantiomers in a synthetic sequence without chiral HPLC control is a common source of batch failure in process development.
| Attribute | BOC-L-Proline | Fmoc-L-Proline | Cbz-L-Proline |
|---|---|---|---|
| Molecular weight | 215.25 g/mol | 337.37 g/mol | 249.26 g/mol |
| N-protecting group lability | Acid-labile | Base-labile | Hydrogenolysis-labile |
| Typical deprotection reagent | TFA or HCl/dioxane | Piperidine | H2/Pd-C |
| Orthogonal stability | Stable to piperidine and H2/Pd-C | Stable to TFA | Stable to piperidine |
| Preferred SPPS mode | Boc/benzyl SPPS | Fmoc/t-Bu SPPS | Not standard SPPS |
On scale, BOC-L-Proline is charged as a crystalline solid through a nitrogen-blanketed split valve into reactors containing dry DMF or dichloromethane. The free carboxylic acid is stable in solution at 20 °C for at least 12 h, but coupling reagents should not be combined in the same charging operation. The preferred sequence in a 50 L glass-lined reactor is to pre-dissolve BOC-L-Proline in DMF, cool to 0–5 °C, add 1-hydroxybenzotriazole and a carbodiimide, and age the activated species for 5–10 min before adding the amine. This pre-activation avoids competitive acylation of the HOBt carboxylate and maintains the pH below neutral. Batch-to-batch variance in residual water and particle size causes slight differences in dissolution rate; larger crystals from recrystallization in ethyl acetate dissolve more slowly than milled material but exhibit lower residual solvent. For automated peptide synthesizers, dissolution in DMF at 0.1 M is standard, with N-methylmorpholine or DIPEA used to neutralize the released HOBt. Pump lines must be flushed with dry DMF after each cycle because BOC-L-Proline solutions in DMF slowly darken when exposed to oxygen and light over 8 h; the resulting discoloration is not necessarily indicative of assay loss but may affect UV monitoring at 214 nm.
Under anhydrous acidolysis, BOC-L-Proline deprotection rate is governed by acid strength, solvent, and temperature. In 50% TFA in dichloromethane at 20 °C, complete removal is generally achieved within 10–30 min, whereas 4 M HCl in dioxane at 0 °C requires 30–60 min; however, published data for this specific configuration is limited, and lot-specific residual water prolongs the terminal stage by competing with carbocation scavenging. Deprotection releases tert-butyl cation, which is scavenged by anisole, thiophenol, or triisopropylsilane; omission of scavengers under high TFA concentration leads to irreversible alkylation of the liberated proline nitrogen. For BOC-L-Proline specifically, the proline carboxyl group remains protonated under acidic conditions, which stabilizes the intermediate oxazolidinone and slows racemization; chiral HPLC after workup typically shows enantiomeric excess above 98%. This is a key difference from less hindered amino acids, where complete racemization can occur in hot TFA. In process development, in-process control by thin-layer chromatography with ninhydrin staining is used to confirm absence of residual BOC-protected starting material before coupling the next amino acid.
