| HS Code | 386979 |
| Product Name | Fmoc-L-alanine |
| Cas Number | 35661-39-3 |
| Molecular Formula | C18H17NO4 |
| Molecular Weight | 311.34 g/mol |
| Synonyms | N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-alanine; Fmoc-Ala-OH |
| Smiles | CC(C(=O)O)NC(=O)OCC1c2ccccc2-c3ccccc13 |
| Appearance | White to off-white powder |
| Purity | ≥98% (HPLC) |
| Melting Point | 148-153 °C |
| Optical Rotation | [α]D20 = -18.5° (c=1 in DMF) |
| Solubility | Soluble in DMF and DMSO; sparingly soluble in ethanol; insoluble in water |
| Storage Conditions | Store at 2-8 °C, protected from light and moisture |
As an accredited Fmoc-L-alanine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Fmoc-L-alanine is supplied as a white powder in a sealed glass bottle, with quantities available as 5 g or 25 g. |
| Container Loading (20′ FCL) | Fmoc-L-alanine packed in drums/pails, loaded into a 20-foot FCL container, secured and sealed for safe transport. |
| Shipping | Fmoc-L-alanine ships at ambient temperature in a sealed, light-protected container to maintain purity. It is not classified as dangerous goods for routine transport. Keep away from moisture and strong oxidizers during transit. Upon receipt, store refrigerated, tightly sealed, and protected from light to ensure stability. |
| Storage | Store Fmoc-L-alanine in a tightly sealed container at –20°C, protected from light and moisture. Ensure the container is desiccated and allowed to warm to room temperature before opening to prevent condensation. Under these conditions, the compound remains stable for long-term use. Avoid repeated freeze-thaw cycles. |
| Shelf Life | Fmoc-L-alanine has a shelf life of 2–3 years when stored dry, cool, and protected from light. |
The incorporation of Fmoc-L-alanine into medium-length peptide APIs begins with its activation as a 1:1:1 DIC/Oxyma mixture and feeding at 3.0–4.0 equivalents relative to the free N-terminal amine on Rink amide AM resin with substitution of 0.3–0.6 mmol/g. The protected amino acid is dissolved in DMF or NMP at 0.1–0.2 M and delivered through a solvent-resistant pump loop; deprotection uses 20% piperidine in DMF for 2 × 10 min, with the dibenzofulvene-piperidine adduct monitored by absorbance at 301 nm. Coupling is held at 20–25 °C for 30–50 min in a jacketed glass vessel with overhead stirring, or at 45–50 °C for 5–10 min in a microwave peptide synthesizer with fiber-optic temperature feedback. Compliance for the resulting peptide API is anchored to ICH Q7 Section 11.4 for laboratory controls, Ph. Eur. 2.2.44 for HPLC purity, USP <621> for system suitability, and ICH Q6B for product-related impurities. The downstream process after chain assembly includes TFA/TIS/water 95:2.5:2.5 cleavage for 2–3 h, precipitation in cold MTBE at −20 °C, preparative C18 HPLC at 10 µm particle size, and counterion exchange to acetate before lyophilization at 0.08 mbar and −45 °C. Terminal product types originating from this route include linear and lactam-bridged therapeutic peptides, GLP-1 receptor agonist generic intermediates, and synthetic peptide hormone drug substances.
| Resin type | Substitution | Fmoc-L-alanine feed | Activator system | Coupling time/temperature |
|---|---|---|---|---|
| Rink amide AM | 0.3–0.6 mmol/g | 3.0–4.0 equiv | DIC/Oxyma 1:1:1 | 30–50 min at 20–25 °C |
| Wang resin | 0.4–0.8 mmol/g | 3.0 equiv | HBTU/DIPEA | 45 min at 25 °C |
| 2-Chlorotrityl chloride | 0.8–1.0 mmol/g | 3.5 equiv | DIPEA in DCM | 60 min at 20 °C |
Within GMP peptide CRO suites running parallel synthesizer banks at 2–50 mmol scale, Fmoc-L-alanine is introduced under ICH Q7 Section 7.1 material management and EU GMP Part II with a batch-record feed of 3.0 equivalents for standard alanine positions and 4.0 equivalents when the preceding residue is N-methylated or part of a turn-inducing motif. Incoming lots are quarantined and tested for identity by FTIR and LC-MS, enantiomeric purity by chiral HPLC with a specification of D-alanine ≤0.5%, residual solvent by headspace GC according to ICH Q3C, and water content by Karl Fischer titration at ≤0.3% w/w. Automated synthesizers with PTFE-fritted reaction vessels alternate DMF and NMP washes, 20% piperidine deprotection, and Kaiser or TNBS tests after each coupling; if free amine remains above 0.1% of theoretical substitution, a recoupling cycle with 2.0 equivalents of fresh Fmoc-L-alanine is triggered. Purification includes preparative RP-HPLC at 215 nm, fraction pooling by UPLC, and lyophilization. Terminal product types include custom research-grade peptides of 5–80 residues, phosphopeptides, biotinylated probes, dye-labeled substrates, and toxicology batch intermediates. A production constraint appears when DMF-swollen resin volume exceeds the capacity of overhead-agitated reactors; static-bed nitrogen-bubbled reactors become necessary because overhead agitation causes resin bead fragmentation and increased backpressure in downstream filtration.
Peptide vaccine candidates containing an alanine residue at position 2 of an MHC class I-binding epitope require low-loading resin conditions and a higher protected amino acid feed to maintain coupling yield across the shortened 9-mer chain. Fmoc-L-alanine is therefore coupled to Wang or 2-chlorotrityl chloride resin at 0.2–0.4 mmol/g using 4.0 equivalents relative to resin substitution, with a DIC/Oxyma preactivation time of 3–5 min in DMF. Manufacturing controls for parenteral vaccine peptide intermediates follow Ph. Eur. 2.6.14 for bacterial endotoxin, ICH Q6B for peptide impurity profiles, and WHO good manufacturing practices for biological products; when the epitope is conjugated to KLH or CRM197, the unconjugated intermediate is also characterized by size-exclusion HPLC to ensure conjugation-competent monomer content. The upstream route uses 20% hexafluoroisopropanol in DCM for cleavage from 2-chlorotrityl resin to preserve side-chain protecting groups, while Rink amide-based vaccine candidates are cleaved with TFA/TIS/water 95:2.5:2.5 for 2 h. Preparative C18 HPLC with 0.1% TFA mobile phases is followed by strong anion-exchange conversion to acetate and lyophilization. Terminal product types include synthetic long peptide cancer vaccine intermediates, palmitoylated T-cell epitope lipopeptides, and peptide–carrier protein conjugates for infectious disease candidates. Endotoxin load in the final peptide is controlled to not more than 5 EU/mg for early toxicology, and lyophilization is the final step because polysorbate 80 in formulation is incompatible with residual TFA above 0.05% w/w.
When an alanine spacer is required immediately C-terminal to a maleimidocaproyl group in a cathepsin-cleavable antibody-drug conjugate linker, Fmoc-L-alanine is loaded as the first Fmoc-protected residue on 2-chlorotrityl chloride resin to permit acid-labile release of protected linker intermediates. The first coupling proceeds in DCM with 3.5 equivalents of Fmoc-L-alanine and 2.0 equivalents DIPEA relative to resin substitution, followed by methanol capping of unreacted chloride sites. ADC linker intermediates are governed by ICH Q7 for API starting materials and ICH M7 for mutagenic impurity assessment; LC-MS monitoring of the Fmoc-Ala-loaded intermediate is performed at the expected [M+H]+ ion, and residual piperidine from Fmoc removal is controlled by GC-MS before subsequent Val-Cit-PAB assembly. After Fmoc deprotection with 20% piperidine, the valine-citrulline-PAB module is assembled using HATU/DIPEA couplings at 0.3 M, and the protected linker is released with 20% trifluoroethanol in DCM rather than TFA to retain the PAB carbamate and alanine spacer. Terminal product classes include Val-Cit-PAB-MMAE, Val-Ala-PAB-SN-38, and other protease-cleavable linker-drug intermediates for ADC development. The initial Fmoc-L-alanine loading on 2-chlorotrityl chloride resin is carried out in DCM with controlled agitation because the acid-labile handle undergoes premature cleavage at pH above 8.5.
The release of a cosmetic peptide for leave-on formulation requires replacement of the TFA counterion with acetate before downstream blending, and Fmoc-L-alanine-derived peptide sequences are manufactured under the same documentation and antiseptic controls as other cosmetic actives. Fmoc-L-alanine is used in three- to ten-residue biomimetic sequences assembled on Rink amide AM resin at 0.5–0.7 mmol/g, with a feed of 3.0 equivalents and DIC/Oxyma activation at 0.1 M in DMF. Process controls align with EC 1223/2009 Article 18 safety assessment, ISO 22716:2007 for cosmetic GMP, and ICH Q3C for residual solvent limits. Cleavage with TFA/TIS/water 95:2.5:2.5 is followed by precipitation in cold diethyl ether, preparative HPLC, and strong anion-exchange treatment on quaternary ammonium resin to generate the acetate salt; the eluate is filtered through 0.22 µm PES, filled into 10 mL lyophilization vials, and dried at −45 °C for 48 h. Terminal product types include palmitoyl tripeptide intermediates, acetyl hexapeptide analogs, and short-chain peptide cosmeceutical candidates containing an alanine residue. Prolonged storage of Fmoc-L-alanine in DMF above 25 °C is avoided because slow Fmoc deprotection and diketopiperazine formation reduce the yield of the desired full-length sequence.
In antimicrobial peptide synthesis at 1–10 mmol scale, sequence-dependent aggregation often appears when three or more alanine residues are positioned consecutively, requiring a differentiated double-coupling rule for Fmoc-L-alanine. Standard coupling employs 3.0 equivalents of Fmoc-L-alanine with HBTU/DIPEA in 0.2 M NMP; for Ala-Ala stretches, a second treatment with 2.0 equivalents and fresh HBTU is run for 45 min before Kaiser testing. Research organizations apply ISO 9001 quality records and release the purified peptide by RP-HPLC at 215 nm and LC-MS; antimicrobial activity of the terminal peptide is evaluated under CLSI M07-A10 broth microdilution and CLSI M100 interpretive criteria. Microwave-assisted synthesis at 50 °C and 20–35 W for 4 min is used when the sequence does not contain temperature-sensitive cysteine branching; cleavage with TFA/thioanisole/water 90:5:5 for 3 h, precipitation in cold diethyl ether at −20 °C, preparative HPLC, and lyophilization follow. Terminal product classes include linear α-helical antimicrobial peptide research lots, synthetic lipopeptide candidates, and truncated analogs for structure-activity studies. Sodium salt content above 0.5% w/w from phosphate-buffered lyophilization depressed observed MIC values in broth dilution assays, so final desalting against 0.1 M ammonium acetate is required before biological evaluation.
Retaining the Fmoc group as an aromatic stacking motif changes the resin-cleavage strategy for peptide amphiphile gelators, and Fmoc-L-alanine is used here to synthesize Fmoc-dipeptide and Fmoc-tripeptide building blocks rather than to generate a final deprotected peptide API. Fmoc-L-alanine is coupled to resin-bound alanine or glycine at 2.0–3.0 equivalents relative to resin loading on 2-chlorotrityl chloride resin at 0.8–1.0 mmol/g, and the product is released with 1–2% TFA in DCM to preserve the N-terminal Fmoc cap. Hydrogel scaffolds intended for cell-contact applications are assessed under ISO 10993-5:2009 for cytotoxicity, ISO 10993-12:2021 for extraction conditions, and USP <71> for sterility when packaged for tissue-engineering use; gelator identity is confirmed by ¹H NMR and LC-MS, with residual DMF controlled to ≤0.1% w/w. The downstream process disperses the Fmoc-dipeptide in water at 0.5–1.0% w/v, adjusts the dispersion to pH 8.5–9.0 with 1 M NaOH, and then forms a self-supporting gel by slow acidification to pH 4.0–5.0 with 0.1 M HCl at 25 °C. Terminal product types include low-molecular-weight peptide hydrogels, extracellular matrix mimetic coatings, and injectable cell culture substrates. Gelation is quenched by phosphate-buffered saline above 50 mM; carbonate-free water is required during pH adjustment to avoid precipitation of insoluble calcium and magnesium carbonates.
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N-α-(9-fluorenylmethoxycarbonyl)-L-alanine, listed in supplier catalogs as Fmoc-Ala-OH and identified by CAS 35661-39-3, is an N-terminal protected chiral amino acid used in Fmoc-based solid-phase peptide synthesis. The compound has an empirical formula C18H17NO4 and a molecular mass of 311.33 g mol−1. The material is supplied as a white to off-white crystalline powder with a melting range of 150–155 °C under USP 741, and the specific optical rotation is controlled between −18.0° and −20.0° at 20 °C for a 1% w/v solution in DMF. HPLC purity is typically specified at ≥99.0% by area normalization at 220 nm. The D-enantiomer content is limited to ≤0.20% because Fmoc-D-alanine has identical molecular mass and nearly identical particulate behavior but generates the opposite configuration in the final peptide.
Water content is limited to ≤0.50% by Karl Fischer titration according to USP 921; residual moisture shortens the shelf life of the activated species and can promote diketopiperazine formation at dipeptide stages. Storage is specified at 2–8 °C in airtight containers protected from light, because the fluorenylmethoxycarbonyl chromophore is sensitive to prolonged exposure at 290–320 nm. The powder is freely soluble in DMF and N-methyl-2-pyrrolidone at 0.1 M, and sparingly soluble in water below pH 7. On automated synthesizers of 1–20 mmol scale, the material is dispensed by gravimetric or volumetric solid-handling modules; caking has been reported when containers are opened under relative humidity above 60% or stored beyond 24 months.
| Parameter | Acceptance range | Method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection |
| Assay | ≥99.0% | HPLC area normalization at 220 nm |
| Specific rotation | −18.0° to −20.0° | 1% in DMF at 20 °C |
| Melting range | 150–155 °C | USP 741 |
| Water content | ≤0.50% | Karl Fischer USP 921 |
| D-enantiomer | ≤0.20% | Chiral HPLC |
| Residual solvents | USP 467 | Headspace GC |
Commercially available Fmoc-L-alanine is offered in at least three specification classes. Research-grade material is typically controlled at ≥98.0% HPLC purity and is suited for exploratory peptide synthesis. Peptide-synthesis-grade material usually adds a minimum assay of ≥99.0%, a water limit of ≤0.50%, and a D-enantiomer limit of ≤0.30%. GMP-grade material is released with full batch documentation, residual solvent data under USP 467, elemental impurity data under ICH Q3D, and stability data supporting retest dating. The choice among these classes is not only a purity question; it determines whether the material can be used in a validated process under ICH Q7 without additional vendor qualification.
In Fmoc SPPS, Fmoc-L-alanine is usually activated in situ with an aminium or carbodiimide reagent. A typical cycle on a 0.1 mmol resin bed uses 3.0–4.0 equiv Fmoc-L-alanine, 3.0–4.0 equiv HATU, and 6.0–8.0 equiv DIPEA in DMF for 30–45 min at 20–25 °C. The methyl side chain of alanine creates only a limited steric shield, so the acylation rate is high; however, the low steric demand permits oxazolone formation under excess base, which is the principal racemization pathway. When pre-activation is performed with 0.9–1.0 equiv DIPEA relative to carboxylate or when OxymaPure/DIC is used, the D-enantiomer in the final peptide can be held below 0.10% by chiral amino acid analysis. In microwave-assisted SPPS at 70 °C, coupling times can be shortened to 5 min, but the racemization rate coefficient increases with temperature; the process window is therefore held at ≤75 °C unless the sequence lacks base-sensitive and chiral-integrity-critical residues. Double coupling or a 5 min bromophenol blue monitoring cycle is applied to poorly accessible resin-bound amines, although alanine itself is not usually the rate-limiting monomer.
On a 10 mmol scale packed-bed synthesizer with a column of 20 cm bed height, the coupling solution residence time is limited by linear flow rate. For Fmoc-L-alanine activated with HATU, recirculation at 2–4 mL min−1 for 30 min typically achieves a negative Kaiser test. If the resin is a high-loading Wang resin above 0.8 mmol g−1, double coupling is used to overcome site accessibility limits. This is less an inherent property of Fmoc-L-alanine than a consequence of resin swelling and the low steric hindrance of alanine, which can form transient oxazolone in the presence of excess base.
Fmoc-L-alanine differs from Boc-L-alanine in removal chemistry rather than in side-chain reactivity. The Fmoc group is removed with 20% piperidine in DMF in 5–10 min, while the Boc group requires 95% TFA or 4 M HCl in dioxane for 30 min or longer. This makes Fmoc-L-alanine compatible with acid-labile side-chain protecting groups such as trityl, tert-butyl, and 2-chlorotrityl linkers. Conversely, Fmoc SPPS is unsuitable for sequences that contain base-sensitive thioesters or sulfamates because repeated piperidine exposure causes degradation. Cbz-L-alanine is deprotected by hydrogenolysis over palladium or by strong acid; its removal is slower and less compatible with automated Fmoc cycles. Fmoc-D-alanine is the enantiomer with identical mass and very similar solubility, but its presence alters peptide conformation and biological recognition; therefore chiral purity is a release criterion. Fmoc-β-alanine is a constitutional isomer with the protected amino group on the β-carbon, and its incorporation adds an additional methylene unit to the peptide backbone, changing amide spacing and protease susceptibility. These materials are not interchangeable in a validated SPPS process unless the downstream analytical methods and impurity profiles are re-qualified.
The resin and linker determine the lower limit of piperidine exposure during N-terminal Fmoc removal. On Wang resin, a 20% piperidine in DMF solution is applied for 5–10 min; on 2-chlorotrityl chloride resin with loading at ≥0.8 mmol g−1, the same exposure can prematurely cleave peptide if the temperature exceeds 25 °C. For such systems, deprotection is often reduced to 2×3 min at 20 °C. Fmoc-L-alanine itself is stable under these conditions, but the deprotection step produces dibenzofulvene-piperidine adduct. If the adduct is not removed by adequate washing, it can alkylate the newly liberated amine and generate truncated or modified sequences. On packed-bed synthesizers, 5–7 column volumes of DMF are typically used after deprotection, and the effluent is monitored by UV at 301 nm to confirm fulvene removal. In stirred-bed reactors, gas agitation below 0.5 L min−1 and resin bed heights above 15 cm have been associated with channeling, residual piperidine pockets, and elevated epimerization of the next Fmoc-L-alanine coupling to 0.25% or higher in the crude peptide. Published data for reactor-specific mixing limits is limited, but the correlation between residual piperidine and epimerization is consistently documented.
| Building block | Removal reagent | Typical time | Compatibility boundary |
|---|---|---|---|
| Fmoc-L-alanine | 20% piperidine in DMF | 5–10 min | Base-labile; avoid thioesters |
| Boc-L-alanine | 95% TFA | 30–60 min | Acid-labile; avoid trityl/2-chlorotrityl |
| Cbz-L-alanine | H2/Pd or HBr/AcOH | variable | Hydrogenation; incompatible with reducible groups |
For GMP-grade Fmoc-L-alanine, the certificate of analysis includes residual solvents from the final crystallization. USP 467 limits DMF as a Class 2 solvent to ≤880 ppm under the default option, and Class 3 solvents such as ethyl acetate are typically controlled below 0.5% w/w. Elemental impurities are assessed under ICH Q3D; if palladium-based hydrogenation or nickel catalysts are used in the route, acceptance criteria of ≤10 ppm Pd and ≤20 ppm Ni are common. The older heavy metal test USP 231 is being replaced by quantitative ICP-MS methods because the colorimetric sulfide method cannot discriminate individual metals at pharmacopeial thresholds. Identity is confirmed by infrared spectroscopy against a qualified reference lot. A change in recrystallization solvent from ethyl acetate/heptane to DMF/water can alter bulk density, particle size distribution, and solid-handling behavior without changing chromatographic purity; such changes require qualification of the automated dispensing process.
Release and in-process control rely on orthogonal methods because Fmoc-L-alanine and its common impurities are not resolved by a single technique. Reverse-phase HPLC at 220 nm quantifies organic impurities; chiral HPLC on a polysaccharide-based chiral stationary phase separates the D-enantiomer; Karl Fischer titration quantifies water; and headspace GC quantifies residual solvents. Identity is confirmed by infrared spectroscopy. Incoming raw material should be tested against certificate-of-analysis values because shipping conditions, especially condensation during cold-chain transfer, can raise water content above 0.50% without a visible change.
Forced degradation studies show that the Fmoc group undergoes β-elimination to dibenzofulvene when exposed to secondary amines or when heated in DMF above 40 °C. The released alanine can then form diketopiperazines at dipeptide stages. The principal impurities observed in aged material are Fmoc-D-alanine, Fmoc-β-alanine, and dibenzofulvene-alanine adducts. Limits for unspecified impurities are commonly set at ≤0.10% and total impurities at ≤0.50% for GMP-grade product. These values are derived from peak area normalization at 220 nm, but they should be supplemented with mass balance data when the material is used in registration batches.
Thermogravimetric analysis shows no significant mass loss below 150 °C in dry nitrogen, but the material should not be dried above 40 °C for extended periods because the Fmoc group undergoes thermal β-elimination. The recommended drying condition is vacuum drying at 25–35 °C for 12–24 h when Karl Fischer water is above 0.50%. In solution, Fmoc-L-alanine in DMF is best used within 24 h when stored at 2–8 °C; protection from light is necessary because the fluorenyl chromophore undergoes photolysis at 300 nm. These boundaries are most relevant in multi-day GMP campaigns where a single lot may be repeatedly dispensed across 20–30 coupling cycles.
Solvent quality in the coupling solution also affects rate and impurity profile. DMF with water above 0.10% or amine contamination above 50 ppm can consume the activated ester and reduce coupling yield. Solvent drying over molecular sieves with 3 Å pore size is used, but Fmoc-L-alanine should not be pre-dissolved in DMF containing excess piperidine or morpholine because premature Fmoc removal occurs. In large-scale peptide synthesizers, the solution is filtered through a 0.45 μm or 0.22 μm membrane before transfer to the reactor to remove insoluble oligomers and dust.
