| HS Code | 222101 |
| Product Name | Fmoc-L-His(Trt)-OH Pharma Grade API |
| Chemical Name | N-alpha-Fmoc-N-im-trityl-L-histidine |
| Synonyms | Fmoc-His(Trt)-OH; N-alpha-Fmoc-N-im-trityl-L-histidine; Fmoc-L-His(Trt)-OH |
| Cas Number | 109425-51-6 |
| Molecular Formula | C40H33N3O4 |
| Appearance | White to off-white powder |
| Purity | >=98.0% (HPLC) |
| Grade | Pharma Grade API |
| Storage | Store at 2-8 deg C, protected from light and moisture |
| Solubility | Soluble in DMF, DCM, DMSO; practically insoluble in water |
| Dosage Forms | Tablet, Capsule, Granule, Injection |
| Route Of Administration | Oral, Injectable |
| Use | Intermediate for peptide synthesis in pharmaceutical APIs |
| Water Content | <=1.0% (Karl Fischer) |
| Heavy Metals | <=20 ppm |
| Residue On Ignition | <=0.1% |
| Loss On Drying | <=1.0% |
| Shelf Life | 24 months |
| Packaging | 1 kg, 5 kg, 10 kg, 25 kg |
| Iupac Name | (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-[1-(triphenylmethyl)-1H-imidazol-4-yl]propanoic acid |
As an accredited Fmoc-L-His(Trt)-OH Pharma Grade API for Tablet / Capsule / Granule / Injection, Oral & Injectable factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
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Fmoc-L-His(Trt)-OH is a protected histidine building block for solid-phase peptide synthesis. In pharmaceutical peptide API manufacturing it is not tableted, encapsulated, granulated, or injected directly. Its downstream use occurs inside automated SPPS reactors where N-terminal histidine residues are installed. The dosage forms specified for the product line are therefore reached through peptide drug substances that contain histidine residues, not through direct formulation of the protected amino acid. For GLP-1 receptor agonist backbones, the histidine residue is usually coupled onto a Rink amide or Wang resin with a substitution of 0.2–0.5 mmol/g. The coupling solution is prepared at 3.0–4.0 equivalents of Fmoc-L-His(Trt)-OH relative to resin-bound free amine. Activation with DIC and Oxyma Pure in dry DMF at 20–25°C proceeds for 60–120 min. Fmoc removal uses 20% piperidine in DMF. Cleavage from the resin uses TFA/triisopropylsilane/water at 95:2.5:2.5 v/v/v, which simultaneously removes the trityl side-chain protecting group. The crude peptide is precipitated in cold methyl tert-butyl ether, washed, and purged with nitrogen. Purification is performed by preparative reverse-phase HPLC on C18 or C8 silica with acetonitrile/water gradients containing 0.1% TFA. Salt exchange to acetate is performed by preparative HPLC. The resulting peptide API, such as a semaglutide backbone, is then lyophilized or formulated into an injectable finished dosage form. Automated synthesizers in commercial campaigns range from 50 mmol laboratory instruments to 1–5 mol PTFE-lined stirred reactors. The Fmoc deprotection step is monitored by UV absorbance at 301 nm to confirm release of the fluorenylmethoxycarbonyl group. Coupling completion is verified by Kaiser or TNBS testing. Raw material lot-to-lot variation in residual DMF, trityl alcohol content, and enantiomeric purity is a recognized source of coupling yield drift in multi-kilogram peptide campaigns.
Racemization control for Fmoc-L-His(Trt)-OH depends on activation chemistry and the trityl side-chain protective group. The DIC/Oxyma Pure system is used at a 1:1 molar ratio with the protected amino acid because it suppresses racemization without generating the byproducts associated with HOBt. HOBt hydrate introduces crystalline water that hydrolyzes the active ester; for moisture-sensitive Fmoc-L-His(Trt)-OH coupling, this water can reduce conversion by a measurable amount when DMF water content exceeds 0.05%. The coupling temperature is maintained at 20–25°C to balance reaction rate and racemization. At 0.30 mmol/g resin loading, a 3.0 equivalent charge of Fmoc-L-His(Trt)-OH in DMF provides a process window that accommodates resin hydration and equipment dead volume. For sequences containing β-branched residues adjacent to histidine, two consecutive couplings of 3.0 equivalents each are common. COMU with DIPEA may be substituted when the peptide sequence shows slow acylation. Chiral HPLC is used to control D-histidine content. Process developers typically set an acceptance criterion of not more than 0.1 area percent for the D-enantiomer, although published data specific to Fmoc-L-His(Trt)-OH racemization on automated synthesizers is limited. The trityl protecting group prevents δ-N acylation during the coupling step; incomplete trityl removal during acid cleavage is avoided by adding triisopropylsilane as a carbocation scavenger at 2.5% v/v. The resulting unprotected histidine residue can then participate in the intended peptide structure. Each commercial peptide campaign qualifies the protected histidine raw material on the actual synthesizer and resin lot to avoid process drift.
After SPPS and peptide purification, residual triphenylmethanol from the trityl protecting group is the most relevant histidine-related impurity in injectable peptide APIs. The crude cleavage mixture contains triphenylmethanol and trityl-containing side products. Precipitation in cold methyl tert-butyl ether removes most of the tritylated mass; preparative HPLC removes the remainder. Final LC-MS analysis typically controls triphenylmethanol at a level consistent with ICH Q3A thresholds for unspecified impurities. The peptide API is concentrated as an acetate salt and lyophilized with mannitol or trehalose as a bulking agent. Lyophilization cycles for histidine-containing peptides use a primary drying shelf temperature between -25°C and -10°C and a chamber pressure of 50–150 mTorr. Collapse of the cake is a recognized failure mode if the product temperature exceeds the collapse temperature. The residual moisture specification for the lyophilized drug product is generally below 1.0% w/w by Karl Fischer titration. The finished injection is released under 21 CFR 211 with bacterial endotoxin limits per USP <85> and particulate matter limits per USP <788>. The protected histidine raw material is not directly mentioned in the finished product dossier, but its residual solvents and elemental impurities are captured through the peptide API release under ICH Q3C and ICH Q3D. Manufacturing experience shows that residual DMF in Fmoc-L-His(Trt)-OH must be controlled before coupling because the solvent is carried into the crude peptide and can affect subsequent lyophilization solvent screening. A typical release specification for residual DMF in the protected amino acid is aligned with ICH Q3C Option 1; the actual limit is peptide campaign-specific.
For oral peptide dosage forms, Fmoc-L-His(Trt)-OH influences the finished tablet or capsule only through the purity and molecular integrity of the peptide API. In the oral semaglutide-type tablet route, the peptide API is formulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl)amino]caprylate, also called SNAC, in a solid matrix. The tablet manufacturing process typically includes dry blending, roller compaction or wet granulation, and tablet compression at main compression forces that produce a hardness range of 60–120 N depending on tooling and tablet size. The protected histidine raw material is not present in the granule or tablet blend; therefore direct compatibility with SNAC is not a specification for Fmoc-L-His(Trt)-OH. What the downstream formulator controls is the peptide API purity profile, which carries forward only those impurities not removed during SPPS and preparative HPLC. Residual triphenylmethanol, dibenzofulvene, and piperidinyl byproducts from histidine introduction are controlled in the peptide API before dry blending. Residual solvent and elemental impurity data for the protected amino acid are evaluated under ICH Q3C and ICH Q3D because these impurities may be removed during SPPS but their input burden affects the API purification load. For capsule development, the peptide API is often spray-dried or lyophilized alone before filling into hydroxypropyl methylcellulose capsules. Granulation of peptide APIs is limited by thermal and moisture sensitivity; at relative humidity above 60%, agglomeration and water uptake can destabilize the peptide and complicate capsule filling. The final oral tablet or capsule is tested for assay, content uniformity, dissolution, and impurities per Ph. Eur. 2.9.3 or USP <711>. The Fmoc-L-His(Trt)-OH material specification therefore focuses on parameters that affect peptide API purity, not on direct excipient compatibility.
Depot injectable poly(lactide-co-glycolide) microsphere formulations are another downstream route for peptides containing N-terminal histidine residues. Exenatide, a 39-amino acid GLP-1 receptor agonist with N-terminal histidine, is formulated as a once-weekly microsphere suspension. The peptide API is synthesized by SPPS using Fmoc-L-His(Trt)-OH for the N-terminal histidine. The purified peptide, as acetate salt, is then encapsulated into PLGA 50:50 or 75:25 microspheres by a double emulsion or coacervation process. In a water-in-oil-in-water double emulsion, the peptide is dissolved in an aqueous phase, emulsified into a polymer solution in methylene chloride, and the primary emulsion is dispersed into an outer aqueous phase containing polyvinyl alcohol. The solvent is evaporated to form microspheres with a drug loading typically in the range of 3–7% depending on the formulation. The microsphere suspension is lyophilized and reconstituted before intramuscular injection. Viscosity of the reconstituted suspension is monitored because particle settling affects syringability. The final product is released under 21 CFR 211 with particulate matter, endotoxin, and peptide purity testing. Residual methylene chloride is controlled under ICH Q3C. For this depot route, the enantiomeric purity and trityl residual profile of Fmoc-L-His(Trt)-OH are upstream inputs that affect encapsulated peptide purity and in vitro release reproducibility. Published data specific to histidine-related impurities in PLGA microsphere release is limited; process developers use the peptide API impurity profile as the primary control point.
Moisture content in Fmoc-L-His(Trt)-OH is a process-critical property for SPPS campaigns that feed capsule-destined peptide APIs. The protected amino acid is stored under dry nitrogen or argon with desiccant. Water content is measured by Karl Fischer titration and is generally controlled below 0.1% w/w for cGMP peptide synthesis. If the material is exposed to ambient relative humidity above 60% during manual dispensing, hydration can deactivate DIC/Oxyma coupling by hydrolyzing the activated carboxylate. The observable effect is a drop in first-coupling conversion that is batch-dependent, often requiring a re-coupling cycle with additional 2.0–3.0 equivalents. For peptide APIs intended for capsules, this affects not only yield but also purification difficulty because truncated or deletion peptides with missed histidine couplings elute close to the target peptide on reversed-phase HPLC. The capsule dosage form then carries the burden of stricter API purity criteria to avoid dosing inaccuracies. Production-scale peptide synthesizers address this issue with dry-box dispensing, closed charging ports, and in-line DMF water monitoring. DMF with water content above 0.03% is considered unsuitable for Fmoc-His(Trt)-OH coupling. For oral capsules, the final peptide API is dried under vacuum at 25–35°C after lyophilization before filling into capsules to prevent moisture-induced degradation and capsule shell brittleness. The release specification for the capsule-filling peptide acetate typically includes water content, purity, and residual solvent tests under ICH Q3C. The operational boundary is clear: unprotected storage or wet dispensing of Fmoc-L-His(Trt)-OH increases process variability; controlled water content is a prerequisite for reproducible coupling and downstream capsule uniformity.
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Fmoc-L-His(Trt)-OH Pharma Grade API for Tablet / Capsule / Granule / Injection, Oral & Injectable is N-α-9-fluorenylmethoxycarbonyl-Nim-trityl-L-histidine, CAS 109789-74-2, molecular formula C40H33N3O4, and molecular weight 619.7 g/mol. The commercial nomenclature also includes Fmoc-His(Trt)-OH and N-alpha-Fmoc-N-im-trityl-L-histidine. The material is supplied as a white to off-white powder and is manufactured under a quality system aligned with ICH Q7 for use as a GMP starting material or intermediate in peptide active pharmaceutical ingredient synthesis. The designation “Pharma Grade API” does not mean that the protected amino acid itself is directly compressed into tablets, filled into capsules, granulated, or injected. Instead, it is consumed during solid-phase peptide synthesis to produce peptide APIs that are later purified, isolated, and formulated into oral or injectable finished dosage forms.
Because the Fmoc group is base-labile and the trityl side-chain protecting group is acid-labile, Fmoc-L-His(Trt)-OH supports an orthogonal protection strategy during peptide chain assembly. The Fmoc group is removed selectively with piperidine in N,N-dimethylformamide, while the trityl protection remains intact until final cleavage. This distinction is critical when histidine residues are incorporated into sequences intended for long-chain peptides or peptide active ingredients requiring multiple acid-labile side-chain deprotection steps.
At relative humidity above 60%, the powder may absorb water and should be equilibrated in a dry nitrogen glovebox before weighing. Contact with primary or secondary amines causes premature Fmoc removal; contact with strong aqueous acid below pH 2 detaches the trityl side-chain protection. Storage is therefore at 2–8°C in a desiccated container under argon, and repeated warming to room temperature should be limited to ≤3 cycles unless stability data for the specific batch support otherwise.
The release specification for pharma-grade Fmoc-L-His(Trt)-OH is defined by organic purity, chiral purity, water content, residual solvents, and trace metal burden. These are controlled because they directly affect downstream peptide API yield, racemization burden, and purification load. Table 1 lists typical acceptance values derived from manufacturer certificates of analysis and pharmacopoeial general methods.
| Attribute | Method | Acceptance criterion |
|---|---|---|
| Appearance | Visual inspection | White to off-white powder |
| HPLC purity | RP-HPLC at 220 nm | ≥99.0% area |
| Chiral purity | Chiral HPLC | ≥99.5% L-isomer |
| Water content | Karl Fischer, Ph. Eur. 2.5.12 | ≤1.0% |
| Residual solvents | GC headspace, USP <467> | ICH Q3C class 2 limits; class 1 solvents absent |
| Heavy metals | Ph. Eur. 2.4.8 | ≤10 ppm |
| Trifluoroacetate | Ion chromatography | ≤0.1% |
| Specific rotation | Polarimetry, 589 nm, c=1 in methanol | −20° to −26° |
On an analytical scale, batch release is performed by reversed-phase HPLC using a 150 mm × 4.6 mm C18 column with 5 µm particles, a flow rate of 1.0 mL/min, and UV detection at 220 nm. The mobile phase consists of 0.1% phosphoric acid in water and acetonitrile. Chiral impurity is quantified on a polysaccharide-based chiral column with n-hexane/2-propanol mobile phase; the D-enantiomer is limited to ≤0.5%. Mass confirmation is performed by LC-MS with electrospray ionization in positive mode; the protonated molecular ion is observed at m/z 620.7. Residual solvent analysis is performed by headspace gas chromatography with flame ionization detection using a 30 m × 0.32 mm DB-624 column and a temperature ramp from 40°C to 240°C at 10°C/min. This resolves dichloromethane, methanol, ethyl acetate, and N,N-dimethylformamide. Moisture content is determined by volumetric Karl Fischer titration per Ph. Eur. 2.5.12 with a drift background below 10 µg/min.
Long-term stability data for this protected amino acid are generated under ICH Q1A storage conditions at 5°C ± 3°C and 25°C ± 2°C/60% RH ± 5% RH. At 25°C/60% RH, water uptake and Fmoc deblocking limit shelf life; the product is therefore stored refrigerated and used within manufacturer-specified retest intervals. Containers are typically high-density polyethylene drums with double polyethylene liners and silica gel desiccant.
The trityl substituent at the imidazole nitrogen exerts steric hindrance and suppresses base-catalyzed racemization during carbodiimide or aminium activation. In Fmoc-L-His-OH without side-chain protection, the imidazole ring can act as an intramolecular base and promote α-proton abstraction, leading to oxazolone formation and D-enantiomer incorporation. The trityl derivative blocks this pathway but slows acylation of hindered secondary amines. In production-scale SPPS reactors with intermittent nitrogen agitation and sintered glass frits, the building block is dissolved in DMF or N-methyl-2-pyrrolidone at 0.10–0.30 M and preactivated with HATU and N,N-diisopropylethylamine at 20–25°C. Coupling recirculation is maintained for 30–120 min. Completion is assessed by the Kaiser test or chloranil test; if residual amine is detected, a second coupling with fresh activated building block is performed. For sequences containing consecutive histidine residues or N-methyl amino acids, double coupling cycles of 2 × 45 min at 25°C with 3 equiv of Fmoc-L-His(Trt)-OH are used. At active ester concentrations above 0.30 M, precipitation can occur in some DMF batches at 20°C; below 0.10 M, coupling rate decreases. Published kinetic data for this exact sterically hindered configuration in every resin type are limited; process development studies are therefore batch-specific.
Following incorporation of Fmoc-L-His(Trt)-OH, the N-terminal Fmoc group is removed by 20% piperidine in DMF for 5–7 min at 20–25°C. The deprotection effluent is monitored at 301 nm; the dibenzofulvene-piperidine adduct has an extinction coefficient of approximately 7,800 M−1 cm−1. This UV-monitored deprotection step is standard in automated peptide synthesizers and allows real-time coupling cycle decisions. Inadequate deprotection leads to deletion peptides that are difficult to remove by preparative HPLC.
Removal of the peptide from the resin and deprotection of the histidine side chain are conducted simultaneously in a mixture of trifluoroacetic acid, triisopropylsilane, and water at 95:2.5:2.5 v/v/v for 2–4 h at 20–25°C. The trityl group is released as a trityl cation. Triisopropylsilane serves as a cation scavenger; insufficient scavenger concentration results in irreversible reattachment of the trityl cation to the imidazole ring or to tryptophan residues if present. In agitated glass reactors with nitrogen blanketing, the cleavage mixture is filtered from the resin and precipitated in cold methyl tert-butyl ether at −20°C to −10°C. Residual trityl alcohol and trityl ethers are then removed by preparative RP-HPLC on C18 silica with 0.1% TFA-modified acetonitrile/water gradients. The target peptide fraction is lyophilized at a shelf temperature of −40°C and a chamber pressure of 50–100 µbar when the final API is an injectable lyophilizate. If the final API is intended for oral solid dosage forms, the same purified fraction may instead be spray-dried or lyophilized to bulk powder.
For a peptide API intended for oral solid dosage manufacturing, Fmoc-L-His(Trt)-OH does not persist into the formulated product. The purified peptide API is concentrated by tangential flow filtration using a 3–10 kDa regenerated cellulose membrane, then lyophilized or spray-dried. For tablets and capsules, the spray-dried peptide API is typically milled and screened through a 60-mesh sieve before blending. Granulation, if required, is performed with a high-shear granulator at impeller speeds of 200–400 rpm and binder addition rates that maintain granule moisture below 5%. Pharmacopoeial tests for the finished dosage form—such as USP <788> for injectable particulate matter, USP <905> for tablet content uniformity, or Ph. Eur. 2.9.19 for particulate contamination—apply to the final peptide product, not to this protected amino acid. Batch records for the protected amino acid support the downstream API dossier but do not replace final product release testing.
When a histidine-containing peptide API is intended for oral solid dosage manufacturing, the protected amino acid remains an upstream input. The critical interface occurs after purification, when the peptide API is isolated as a dried solid. The isolated peptide may be amorphous or partially crystalline depending on lyophilization parameters and residual trifluoroacetic acid content. For tablets, the peptide API is blended with microcrystalline cellulose, mannitol, or dicalcium phosphate diluents; the blend is compressed on a rotary tablet press with precompression force between 1–5 kN and main compression force between 5–20 kN, depending on peptide load and tensile strength target. For capsules, the peptide API is filled into hard gelatin or hypromellose capsules using dosator or tamping-pin equipment; granulation may improve flow if the bulk peptide powder has a Carr index above 25%. For injectable dosage forms, the purified peptide is formulated as a sterile solution or lyophilized cake; compatibility with silicone tubing, stainless steel, and type I glass vials is evaluated under ICH Q1A conditions. Fmoc-L-His(Trt)-OH is not present at this stage and is not tested in these finished dosage forms.
Fmoc-L-His(Trt)-OH differs from Fmoc-L-His(Boc)-OH and Fmoc-L-His-OH in side-chain protection strategy, molecular weight, and cleavage behavior. The trityl derivative has a molecular weight of 619.7 g/mol and is removed by TFA; the Boc derivative has a lower molecular weight of 477.5 g/mol and is also acid-labile but less sterically hindered; the unprotected derivative has a molecular weight of 377.4 g/mol and introduces side-chain nucleophilicity and racemization risk. Table 2 summarizes the technical differences relevant to SPPS process selection.
| Building block | Molecular weight (g/mol) | Side-chain protection | Racemization risk | Cleavage condition | Main use |
|---|---|---|---|---|---|
| Fmoc-L-His(Trt)-OH | 619.7 | Trityl, acid-labile | Suppressed; steric hindrance | TFA/TIS/H2O 95:2.5:2.5 v/v/v | Standard SPPS; long sequences; oral/injectable peptide APIs |
| Fmoc-L-His(Boc)-OH | 477.5 | Boc, acid-labile | Suppressed; less hindered | TFA/scavengers | Sequences requiring less bulky protection |
| Fmoc-L-His-OH | 377.4 | None | Higher; imidazole can act as base | Not applicable | Research use only; avoided in API synthesis |
On 2-chlorotrityl resin, peptide cleavage with 1–2% TFA in dichloromethane at 5–15 min selectively releases protected peptide fragments. Under these conditions, the trityl side-chain group remains substantially intact, whereas a Boc-protected histidine side chain may undergo partial deprotection. This orthogonality is exploited in convergent peptide synthesis and in the production of protected peptide fragments for subsequent fragment condensation. Fmoc-L-His-OH is generally not used in GMP peptide API manufacturing because the unprotected imidazole can produce acylated side products and racemization. The finished dosage form designation in the product title—tablet, capsule, granule, or injection—therefore refers to the final peptide API product route enabled by this building block, not to the direct use of Fmoc-L-His(Trt)-OH as a dosage form ingredient.
