The protected amino acid identified by CAS 71989-26-9, molecular formula C₂₆H₃₂N₂O₆, and molecular mass 468.54 g mol⁻¹ is supplied as Fmoc-L-Lys(Boc)-OH Pharma Grade API. The designation “Pharma Grade API for Tablet / Capsule / Granule / Injection, Oral & Injectable” describes the downstream peptide active pharmaceutical ingredients produced from this starting material; the protected amino acid itself is not milled, granulated, tableted, encapsulated, or injected as a formulated dosage form. Catalog identifiers vary by supplier, and the CAS registry number is the primary product identifier. The molecule carries 9-fluorenylmethoxycarbonyl at the α-amino position and tert-butyloxycarbonyl at the ε-amino position. This orthogonal protection permits selective removal of the α-amine protecting group under basic conditions while the ε-amine remains stable through repeated coupling and washing cycles in solid-phase peptide synthesis. The material is stored at −20°C under desiccant to limit Fmoc β-elimination and moisture uptake. Suppliers release product under GMP quality systems aligned with ICH Q7; each lot is accompanied by a certificate of analysis listing batch-specific results against the specification framework set out below.

How Does Orthogonal Protection in Fmoc-L-Lys(Boc)-OH Affect Solid-Phase Peptide Synthesis?

In Fmoc-based solid-phase synthesis on resins with substitution levels between 0.3 mmol/g and 1.0 mmol/g, the protected lysine is activated with HATU or DIC/Oxyma in DMF or NMP. Coupling protocols in automated peptide synthesizers commonly use 2–4 equivalents of Fmoc-L-Lys(Boc)-OH relative to resin loading, with activator and base excess adjusted to maintain solution pH between 8.0 and 8.5. pH above 9.0 can deprotonate the α-amine after Fmoc removal and reduce coupling efficiency. The Boc ε-amine remains intact under 20% piperidine in DMF, the standard Fmoc removal condition; this prevents ε-amine branching during chain elongation.

At production scale, coupling efficiency of Fmoc-L-Lys(Boc)-OH is sensitive to residual water in DMF and to resin swelling. In a low-pressure SPPS reactor with nitrogen agitation, water contents above 500 ppm in DMF can reduce activation efficiency and increase racemization risk during carbodiimide-mediated coupling. Racemization is controlled by adding Oxyma or HOBt; confirmation uses chiral HPLC after acid hydrolysis and derivatization, with a C18 column and UV detection at 220 nm. Fmoc deprotection is monitored by UV absorbance of the dibenzofulvene–piperidine adduct at 301 nm; incomplete deprotection on commercial synthesizers with UV flow cells is a known source of deletion peptides in lysine-rich sequences. Coupling completion is checked by ninhydrin or chloranil tests; a negative ninhydrin result corresponds to residual free amine below approximately 1% of initial resin sites. If coupling exceeds 30 min without negative ninhydrin, the sequence is flagged for recoupling with fresh activated acid.

Drying of isolated product on a vacuum tray dryer at jacket temperatures between 40°C and 45°C and pressures below 20 mbar reduces residual DMF without thermal Boc deprotection. Thermogravimetric analysis under nitrogen at 10 K min⁻¹ shows no significant mass loss up to 100°C; however, the material is not held at elevated temperatures during processing. Particle-size distribution is controlled by milling and sieving through a 150 µm stainless-steel mesh. Bulk density and tapped density are reported on certificates of analysis according to Ph. Eur. 2.2.42 and USP <616>, because downstream peptide synthesis feed systems may require consistent volumetric dispensing into solid-phase reactors or automated solid-dispensing stations. At 25°C, the compound is freely soluble in DMF, NMP, and DMSO, partially soluble in dichloromethane, and practically insoluble in water; published data for aqueous solubility of this protected amino acid are limited. Aqueous solubility is not a critical process parameter because the protected amino acid is not formulated as an aqueous solution.

Analytical Release and Residual Impurity Control Under ICH Q6A

Each batch is tested against the release specification because impurities in the protected amino acid can carry through to the final peptide API. Related substances are determined by reverse-phase HPLC using a C18 column and UV detection at 220 nm; gradient conditions separate Fmoc-L-Lys-OH, Boc-L-Lys-OH, and diastereomeric or deletion impurities. Integration thresholds are set at 0.05% according to ICH Q3A reporting thresholds for new drug substances. High-resolution mass spectrometry or LC-MS is used to confirm the absence of dimeric impurities, because dimerization produces insertion peptides during solid-phase synthesis.

Residual solvents are measured by headspace GC-FID after dissolving the sample in DMSO; quantification follows ICH Q3C Option 1 with class 2 solvent limits such as DMF ≤880 ppm and dichloromethane ≤600 ppm. Elemental impurities are controlled under ICH Q3D by ICP-MS; for final peptide APIs intended for parenteral use, the permitted daily exposure values for lead, cadmium, arsenic, and mercury are converted into starting material concentration limits based on the manufacturing dilution factor. Published data for this specific configuration are limited where a final dosage form has not been fully developed.

For injectable peptide applications, the starting material is released with bioburden ≤100 CFU/g by Ph. Eur. 2.6.12 and bacterial endotoxins ≤0.25 EU/mg by Ph. Eur. 2.6.14. Final sterile filtration of the peptide solution through a 0.22 µm sterilizing-grade membrane does not remove dissolved endotoxin or residual organic impurities, so controls at the protected-amino-acid stage are mandatory. Analytical methods used for release are validated under ICH Q2(R1) for specificity, linearity, accuracy, precision, and range; HPLC assay linearity is typically established over 80–120% of the nominal test concentration.

Because Fmoc-L-Lys(Boc)-OH batches are produced via N-α-Fmoc protection of L-lysine followed by Boc protection and recrystallization, batch-to-batch variance in residual solvent and crystalline habit may arise from cooling rate in the crystallizer. On a 100 L glass-lined reactor, controlled cooling at 0.5 K min⁻¹ and seed addition at 45°C are used to maintain a consistent crystalline habit. Downstream automated solid-phase synthesizers equipped with gravimetric dispensing hoppers may exhibit bridging when the material contains fines above 20% by laser diffraction; sieving through 150 µm mesh reduces feed interruptions.

The material is incompatible with secondary amines such as piperidine, strong organic bases, and hydrazine, because these reagents remove the Fmoc group and generate dibenzofulvene adducts. Exposure to relative humidity above 60% causes moisture pickup and can reduce coupling efficiency in carbodiimide-mediated activation. Before weighing, the unopened container is equilibrated to ambient temperature to prevent condensation on the powder surface.

When the Protected Amino Acid Must Support Aseptic Injectable Peptide Manufacturing

Final peptide APIs synthesized from Fmoc-L-Lys(Boc)-OH are often purified by preparative reverse-phase HPLC, lyophilized, and sterile-filtered for injection. The side-chain Boc group is removed during cleavage from the resin with TFA/water/triisopropylsilane mixtures, commonly 95:2.5:2.5 v/v/v at 25°C for 2–4 h. tert-Butyl cations formed from the Boc group are scavenged by triisopropylsilane; without scavenger, re-alkylation of tryptophan or cysteine residues may occur. Incomplete Boc deprotection is detected by LC-MS as a mass shift of 100.12 Da; final peptide release specifications commonly limit such impurities to ≤0.5%.

Residual trifluoroacetic acid in the final peptide must be controlled by ion exchange or lyophilization; failure to remove TFA can shift the pH of a reconstituted injectable solution to below 3.0. The final peptide solution is filtered through 0.22 µm PVDF or PES sterilizing-grade filters and filled into Type I borosilicate glass vials under ISO 14644-1 Class 5 conditions. Sub-visible particle testing follows Ph. Eur. 2.9.19; particle counts at ≥10 µm and ≥25 µm are controlled according to harmonized pharmacopoeial limits. Soluble impurities originating from the starting material are not removed by filtration.

Process-mass intensity calculations for SPPS production lines show that protected amino acid input mass may exceed final peptide mass by a factor of 8–15 depending on sequence length and purification yield. This magnification makes starting-material purity a critical control point for injectable peptides, where the final peptide is typically released with individual unknown impurities ≤0.5% and total impurities ≤2.0% under ICH Q6A for new drug substances. The Fmoc-L-Lys(Boc)-OH specification therefore includes strict limits on Fmoc-L-Lys-OH and ε-deprotected lysine derivatives to avoid carryover of des-lysine or insertion impurities.

Although Fmoc-L-Lys(Boc)-OH is not directly tableted or encapsulated, the final peptide API synthesized from it can be formulated into oral granules by low-shear wet granulation or dry granulation. In dry granulation with a roll compactor, granule density is typically controlled between 0.45 g/mL and 0.65 g/mL by roller pressure; the resulting granules are blended with crospovidone and magnesium stearate before tableting. For acid-labile peptide APIs, enteric coating with a methacrylic acid–ethyl acrylate copolymer delays release until pH 5.5 or higher; dissolution is tested according to USP <711> with two-stage media. No Fmoc-L-Lys(Boc)-OH-derived protecting group remains in the final dosage form because Fmoc and Boc groups are removed during solid-phase synthesis and cleavage. Capsule formulations for oral peptides may use enteric-coated hard capsules to protect the peptide from gastric pepsin at pH 1.2.

Differences from other protected lysine derivatives become decisive when a peptide contains multiple lysine residues or requires selective side-chain modification. Fmoc-L-Lys(Boc)-OH is selected when the ε-amine must remain protected throughout Fmoc deprotection cycles and be released only during final acidolytic cleavage. Fmoc-L-Lys(Dde)-OH permits on-resin ε-amine deprotection with 2% hydrazine in DMF, but residual hydrazine must be washed out before subsequent coupling. Fmoc-L-Lys(Mtt)-OH permits on-resin deprotection with 1–2% TFA in dichloromethane, but premature Mtt loss can occur during prolonged acidic washing. The Boc derivative therefore has lower on-resin side-chain lability under Fmoc conditions, but it cannot be selectively removed on-resin without resin cleavage. In Boc SPPS, Boc-L-Lys(Boc)-OH is used instead; it requires hydrogen fluoride or strong acid for final deprotection and is not compatible with Fmoc chemistry. The free lysine mass fraction in Fmoc-L-Lys(Boc)-OH is approximately 31%, which is used to calculate feed ratios in multigram and production-scale syntheses.

The material is stored in sealed double polyethylene bags inside a fiber drum at −20°C; before weighing, the unopened container is equilibrated to ambient temperature to prevent condensation. Exposure to relative humidity above 60% causes moisture pickup and can reduce coupling efficiency in carbodiimide-mediated activation. The product is not sterilized before dispatch; final peptide APIs derived from it are sterilized downstream by filtration or aseptic processing. The protected amino acid should not be stored in contact with secondary amines, hydrazine, or strong organic bases, because these remove the Fmoc group and generate dibenzofulvene adducts. Under ICH Q11, the selection of a GMP starting material is based on the point at which significant structural features of the final drug substance are introduced; Fmoc-L-Lys(Boc)-OH is commonly designated as a GMP starting material for peptide APIs after manufacture according to ICH Q7. Regulatory filings may require the peptide API manufacturer to demonstrate that changing Fmoc-L-Lys(Boc)-OH suppliers does not alter impurity profiles above ICH Q3A thresholds.