| HS Code | 590190 |
| Product Name | Fmoc-L-serine |
| Synonyms | Fmoc-Ser-OH; N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-serine |
| Cas Number | 73724-45-5 |
| Molecular Formula | C18H17NO5 |
| Molecular Weight | 327.33 g/mol |
| Purity | ≥98% (HPLC) |
| Appearance | White to off-white powder |
| Melting Point | 220 °C (decomposition) |
| Optical Rotation | [α]20/D = -6.0° (c=2, DMF) |
| Solubility | Soluble in DMF, DMSO, THF, and methanol; sparingly soluble in water |
| Storage Conditions | Store at 2-8 °C, protected from light and moisture |
| Smiles | c1ccc2c(c1)-c3ccccc3C2COC(=O)N[C@@H](CO)C(=O)O |
As an accredited Fmoc-L-serine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Fmoc-L-serine, 25 g, supplied as a white crystalline powder in a sealed amber glass bottle with tamper-evident cap. |
| Container Loading (20′ FCL) | 20′ FCL: Fmoc-L-serine packed in sealed drums on pallets, secured safely, kept dry, labeled correctly, ready for transport. |
| Shipping | Fmoc-L-serine ships as a dry, crystalline compound in sealed containers with desiccant to prevent moisture uptake. Store refrigerated (2–8°C), protected from light and heat. Transport at ambient temperature is typically acceptable for short transit, ensuring stable purity and intact Fmoc protection. |
| Storage | Store Fmoc-L-serine in a tightly sealed container in a cool, dry place, ideally at 2–8°C or below. Protect from light, moisture, and strong oxidizing agents. Keep away from acids, bases, and heat sources. Use appropriate personal protective equipment when handling. |
| Shelf Life | Store tightly sealed, protected from light and moisture at -20°C. Shelf life: typically 2–3 years from manufacture. |
On automated Fmoc/tBu peptide synthesizers using PEG-polystyrene composite resins with substitution grades from 0.15 mmol/g to 0.25 mmol/g, Fmoc-L-serine is introduced without side-chain protection only when the target sequence is short and subsequent coupling steps do not require aggressive activation. Incoming material is released against HPLC area normalization at 220 nm of at least 98.0%, specific rotation [α]D20 = -11.5±1.0° (c=1, DMF), and residual DMF by headspace GC under USP <467> Procedure A. The dry powder is stored at 2–8°C and is dried under vacuum at 25°C for 12 h if the ambient relative humidity has exceeded 60% during dispensing. A 0.4 M stock solution in DMF is prepared and used within 24 h to limit Fmoc degradation and diketopiperazine formation. Coupling on a batch synthesizer of the CS Bio 136X or PTI Tribute type uses 4.0 equiv Fmoc-L-serine, 3.9 equiv HCTU or HBTU, and 8.0 equiv DIPEA relative to the resin-bound amine. Pre-activation is limited to 2–3 min at 20–25°C before transfer to the resin. The coupling is allowed to proceed for 30–45 min; a blue Kaiser test triggers a second coupling with 2 × 45 min contact. Capping after every coupling is performed with acetic anhydride/pyridine (1:2 v/v) in DMF for 20 min. Fmoc deprotection is performed with 20% piperidine in DMF (v/v) in two cycles of 5 min and 15 min, followed by six DMF washes at 30 s each. The principal process failure is O-acylation of the β-hydroxy group by the incoming activated amino acid, producing a branched side-chain sequence that is detected by LC-MS as a +327.3 Da adduct at the serine position. The risk increases with HOAt-based activators, prolonged pre-activation, and coupling temperatures above 25°C. HOBt-based activation, short contact times, and immediate capping reduce the side reaction. For sequences above approximately 15 residues, or when multiple serine residues are clustered, Fmoc-L-serine is replaced by Fmoc-Ser(tBu)-OH in routine GMP peptide manufacture. The unprotected building block is justified as a raw material under ICH Q11 and is included in cleaning validation under ICH Q7; carryover is monitored by single-ion LC-MS at m/z 328.1 [M+H]+.
| Serine derivative | Side-chain protection | Removal or deprotection condition | Primary failure mode | Typical process position |
|---|---|---|---|---|
| Fmoc-L-serine | none | not applicable | O-acylation, β-elimination under strong base | short peptides, solution phase, first residue on Wang resin |
| Fmoc-Ser(tBu)-OH | tert-butyl ether | TFA/TIS/H2O 95:2.5:2.5 v/v/v for 2–4 h | acidolysis; incompatible with acid-sensitive linkers | routine Fmoc SPPS |
| Fmoc-Ser(Trt)-OH | trityl ether | 1–2% TFA in DCM for 5–15 min | premature detritylation during coupling if acidity is uncontrolled | on-resin selective deprotection |
| Fmoc-Ser[PO(OBzl)OH]-OH | monobenzyl phosphate | TFA/TIS/H2O 95:2.5:2.5 v/v/v for 4 h | β-elimination to dehydroalanine under prolonged piperidine | phosphopeptide synthesis |
Anomeric selectivity is controlled mainly by the C-2 ester of the glycosyl donor and the reaction temperature when Fmoc-L-serine is converted into an O-glycosyl-L-serine building block for glycopeptide solid-phase assembly. Peracetylated galactose or glucose trichloroacetimidate donors are condensed with Fmoc-L-serine allyl ester or p-nitrobenzyl ester in dry dichloromethane at -20°C to 0°C using 0.1–0.3 equiv trimethylsilyl trifluoromethanesulfonate. The donor itself is prepared from the peracetylated sugar and trichloroacetonitrile with DBU at 0°C; its anomeric purity is confirmed by ¹H NMR before the coupling. β-Selective galactosylation is favored by the C-2 acetyl participating group. The crude anomeric ratio is checked by ¹H NMR integration between δ 4.30 and δ 5.70. The β-galactosyl H-1 doublet commonly appears near δ 4.50 with J=8.0 Hz, while the α-isomer H-1 doublet appears near δ 5.20 with J=3.5 Hz. The reaction is quenched with triethylamine, filtered through Celite, and purified by flash chromatography on silica gel with hexane/ethyl acetate gradients. The acetyl protecting groups remain on the sugar through the coupling and are removed later on-resin with hydrazine in methanol or sodium methoxide before global acidolysis. This protects the glycosidic bond during Fmoc SPPS. Final glycopeptide APIs are released by Ph. Eur. 2.2.29 HPLC and USP <621> system suitability. Residual silicon from TMSOTf is assessed under ICH Q3D and measured by inductively coupled plasma mass spectrometry when the peptide is destined for parenteral use. If the donor is a β-D-glucopyranosyl bromide under Koenigs-Knorr conditions, silver triflate or silver carbonate in dichloromethane/toluene at -10°C to 0°C is used; silver salts are removed by precipitation with sodium chloride and filtration. Published yields vary with donor and anomeric ratio; identity is confirmed by high-resolution MS and 13C NMR of the anomeric carbon, which for β-linked products typically falls between δ 100 and δ 105.
When a protected phosphoserine building block is not available in the required batch size, Fmoc-L-serine is converted to Fmoc-L-Ser[PO(OBzl)OH]-OH by phosphoramidite chemistry prior to SPPS. Fmoc-L-serine is dissolved in anhydrous THF/DMF at 0°C and phosphorylated with dibenzyl N,N-diisopropylphosphoramidite in the presence of 1H-tetrazole. After 15–30 min, the intermediate phosphite triester is oxidized with tert-butyl hydroperoxide in decane at -10°C to 0°C. The product is isolated by ethyl acetate extraction and silica gel chromatography; fractions are checked by 31P NMR for the phosphotriester signal. The side-chain-protected building block is stable to standard 20% piperidine in DMF for cumulative exposure up to 10–15 min; prolonged piperidine exposure causes β-elimination to dehydroalanine, which appears in LC-MS as a mass loss of 98 Da relative to the phosphoserine residue. Fmoc-L-serine with free hydroxyl is not coupled directly into phosphopeptide sequences; the conversion is carried out ex-resin to avoid side reactions. On a peptide synthesizer, Fmoc-L-Ser[PO(OBzl)OH]-OH is coupled with 4.0 equiv, HCTU 3.9 equiv, and N-methylmorpholine or DIPEA 8.0 equiv in DMF at 20–25°C. Cleavage from the resin uses TFA/TIS/H2O 95:2.5:2.5 v/v/v for 4 h. Benzyl phosphate groups are removed during acidolysis. Residual dibenzyl phosphate-derived impurities are tracked by single-ion monitoring in the final phosphopeptide. Final phosphopeptides used as kinase substrate standards are released by HPLC and LC-MS/MS. If the route contains hydrogenolysis, residual palladium is controlled under ICH Q3D and measured by ICP-MS after acid digestion.
The β-lactone derived from Fmoc-L-serine is prepared under Mitsunobu activation with diisopropyl azodicarboxylate and triphenylphosphine in THF at -78°C to -40°C. The α-amine remains Fmoc-protected, and the starting material is dried to a water content below 0.1% by Karl Fischer titration before the reaction. The β-lactone is electrophilic at the β-carbon and reacts with thiophenol, benzyl mercaptan, or sodium azide in DMF at 0–4°C. Ring opening by thiophenol yields N-Fmoc-S-phenyl-L-cysteine after aqueous workup; azide opening gives N-Fmoc-β-azido-L-alanine, which is compatible with copper-catalyzed azide-alkyne cycloaddition using CuSO₄/sodium ascorbate systems. The route avoids direct alkylation of the serine hydroxyl and produces side-chain-modified alanine analogs without leaving-group-derived impurities. The β-lactone is moisture-sensitive and is used within 12–24 h after preparation. Batch sizes above 25 g are not routinely reported in public industrial literature; production-scale data for this specific configuration is limited. Analytical control is by ¹H NMR for the β-lactone methine protons and HPLC at 254 nm. Triphenylphosphine oxide is tracked by 31P NMR; residual triphenylphosphine oxide is held below 0.5% by 31P NMR integration in the ring-opened intermediate.
Serine provides a trifunctional architecture for solid-phase dendrimer construction: the Fmoc-bearing α-amine remains protected while the α-carboxyl is anchored to a Rink amide or Wang resin and the β-hydroxyl is acylated as a second elongation point. The β-hydroxyl is activated with DIC/DMAP in DMF/DCM (1:1 v/v) and reacted with an Fmoc-amino acid such as Fmoc-β-alanine or Fmoc-Gly-OH. The acylation is run for 12–16 h at 20–25°C; incomplete esterification is detected by FTIR loss of the hydroxyl stretch at 3300–3500 cm⁻¹ or by analytical TLC after cleavage of a resin aliquot. Piperidine-mediated Fmoc removal must be limited to 5 min cycles because the side-chain ester bond is susceptible to aminolysis with prolonged exposure. After deprotection of both the α-amine and the side-chain ester-linked amine, a two-branch node is generated. Repeating the cycle builds first- and second-generation peptide dendrimers. This application is documented mainly at laboratory scale; preparative HPLC with a 250 × 21.2 mm C18 column, 5 µm particles, and linear 0.1% TFA/acetonitrile gradients from 5% to 45% over 40 min is used to isolate the final dendrimer. Residual DMAP is controlled by LC-MS single-ion monitoring. Fmoc-L-serine with free hydroxyl is preferred over Fmoc-Ser(tBu)-OH in this use because the tBu ether would require TFA treatment that could cleave the resin linker prematurely.
Solution-phase peptide fragment coupling can use Fmoc-L-serine without side-chain protection when the final product is a short dipeptide or tripeptide intermediate and no later carbodiimide-mediated step will leave the β-hydroxyl exposed. The carboxyl group is activated as a mixed anhydride by adding isobutyl chloroformate and N-methylmorpholine in THF at -15°C to 0°C. After 2–3 min, the anhydride is treated with an amino acid tert-butyl ester or glycinamide in ethyl acetate/THF. The reaction is quenched with 5% aqueous citric acid, washed with 5% sodium bicarbonate and brine, and dried over sodium sulfate. The free hydroxyl permits later selective O-acylation or O-phosphorylation of the isolated peptide intermediate. If the hydroxyl is not intended for further derivatization, O-acylated impurities formed during workup are resolved by HPLC and compared against reference standards. Residual solvent control follows ICH Q3C: DMF is maintained below 880 ppm and THF below 720 ppm in the isolated intermediate. The use of Fmoc-L-serine in solution phase reduces the number of acid deprotection steps but introduces sensitivity to base: aqueous sodium carbonate above 10% at elevated temperature can promote β-elimination to dehydroalanine. Fmoc-Ser(tBu)-OH is preferred when the solution-phase route contains a hydrogenation or hydrazine treatment step that would be incompatible with the free hydroxyl.
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Fmoc-L-serine (CAS 73724-45-5; molecular formula C18H17NO5; relative molecular mass 327.34) is the N-α-9-fluorenylmethoxycarbonyl derivative of L-serine in which the α-amine is blocked and the primary side-chain hydroxyl remains free. This structural state separates it from the tert-butyl- and trityl-protected serine derivatives used in standard Fmoc/tBu solid-phase peptide synthesis. The free hydroxyl is stable to the 20% piperidine/DMF solutions used for Fmoc removal, but it introduces a competing oxygen nucleophile during activated-ester coupling. Consequently, Fmoc-L-serine is selected not as a default serine building block but as a deliberate intermediate when a free hydroxyl must be present during assembly, on-resin modification, or final cleavage.
Commercial material is supplied in peptide synthesis grade and cGMP-grade designations; catalog grades differ primarily in residual solvent documentation, endotoxin testing, and packaging environment rather than in the underlying chemical structure. Catalog designations for this material are not fully harmonized. The same molecule appears as Fmoc-L-serine, Fmoc-Ser-OH, and N-α-Fmoc-L-serine. There is currently no harmonized pharmacopeial monograph specific to Fmoc-L-serine; release criteria are supplier-defined and aligned with general chromatographic and residual-solvent chapters such as USP <621>, USP <921>, and USP <467>.
Peptide-synthesis-grade Fmoc-L-serine is typically released against the following matrix. HPLC purity is determined by reversed-phase C18 columns (150 mm × 4.6 mm, 5 µm) with UV detection at 220 nm using a water/acetonitrile/0.1% TFA mobile phase. Chiral purity is measured on an amylose tris(3,5-dimethylphenylcarbamate) stationary phase to quantify the unwanted D-enantiomer. Water content by coulometric Karl Fischer titration is controlled because moisture accelerates slow Fmoc hydrolysis and reduces apparent molar charge when weighing for automated synthesis. Residual solvent profiles are evaluated by headspace gas chromatography against USP <467>.
| Parameter | Analytical method | Typical release limit |
|---|---|---|
| Appearance | Visual inspection | White to off-white crystalline powder |
| Assay (HPLC) | Reversed-phase C18 column, 150 mm × 4.6 mm, 5 µm; 0.1% TFA water/acetonitrile gradient; UV 220 nm | ≥ 98.0% |
| Enantiomeric purity | Chiral HPLC, amylose tris(3,5-dimethylphenylcarbamate) column | ≥ 99.5% L-isomer |
| Water content | Coulometric Karl Fischer titration | ≤ 0.5% |
| Residual solvents | Headspace GC per USP <467> | Class 3 limits |
Chromatographic system suitability is established before lot release. For reversed-phase HPLC, the resolution between Fmoc-L-serine and its nearest Fmoc-protected impurity is set not less than 1.5, the tailing factor not more than 2.0, and the injection-to-injection relative standard deviation not more than 1.0% for six replicate injections. Failure to meet these conditions indicates column aging or mobile-phase premixing error, and the run is invalid for release. Chiral HPLC system suitability uses a racemized or mixed D/L standard to confirm resolution not less than 2.0 before sample quantification.
HPLC purity below 98.0% generally indicates contamination by Fmoc-protected dipeptide, hydrolyzed amino acid, or fluorenylmethyl by-products. Chiral HPLC detection is necessary because Fmoc-L-serine may be contaminated by the D-enantiomer during synthesis or resolution; for peptide active pharmaceutical ingredients, a D-serine impurity above 0.5% can propagate into a diastereomeric peptide that is difficult to remove by preparative HPLC. Moisture above 0.5% is operationally significant because the effect is expressed as lower available molar charge during automated synthesis.
For solution preparation, Fmoc-L-serine is dissolved in DMF or NMP to 0.1–0.2 M, which corresponds to approximately 32–65 mg/mL for the 327.34 molecular mass. Higher concentrations are possible in NMP but may slow complete dissolution. Dichloromethane is not recommended as the primary solvent for this unprotected derivative; if a DCM/DMF mixture is required for resin swelling compatibility, the DMF proportion should remain at least 50%. After dissolution, the solution should be used within 4–6 h at 15–25 °C; longer holding times allow slow Fmoc degradation by residual water and increase the activated-ester side-reaction profile.
In Fmoc/tBu SPPS, standard amino acids are often coupled with 2–4 equivalents of activated amino acid to drive difficult sequences to completion. Fmoc-L-serine does not tolerate this excess without side-chain protection, because the primary hydroxyl can be O-acylated once the resin-bound amine is consumed. The resulting O-acyl adduct is cleaved from resin as a branched peptide and cannot be reversed by piperidine. For this reason, Fmoc-L-serine is coupled with 1.05–1.20 equivalents of activated amino acid and 1.0 equivalent of coupling reagent relative to the activated carboxyl group. Reaction completion is confirmed by Kaiser test or TNBS test; if a recoupling is required, the resin is washed extensively with DMF to remove residual active ester before a second low-excess coupling is attempted.
Coupling with DIC/Oxyma is performed by pre-activating Fmoc-L-serine for 5–10 min at 0–5 °C in DMF. The resulting oxime ester is less reactive toward the serine hydroxyl than the corresponding HOBt ester, but it is not inert. Aminium reagents such as HBTU and HATU generate more electrophilic species and are used only when the free hydroxyl is not present or when the sequence is short. If HBTU is used, the Fmoc-L-serine solution is added to the resin immediately after 1 min of activation, and the reagent excess is limited to 1.0 equivalent relative to the carboxyl group.
Fmoc removal from the N-terminal serine residue after coupling is accomplished with 20% piperidine in DMF for 2 × 5 min at room temperature or 1 × 20 min for difficult sequences. The free side-chain hydroxyl is stable to piperidine, but Fmoc deprotection solution should be drained and washed with DMF until the UV trace at 301 nm returns to baseline; residual dibenzofulvene-piperidine adduct can otherwise absorb into the resin and complicate subsequent chloranil or Kaiser testing.
Capping after coupling with acetic anhydride/pyridine is not recommended when Fmoc-L-serine is already present as the side-chain-unprotected residue, because acetylation of the unprotected hydroxyl can occur under standard capping conditions. If capping is necessary, a short 2 min exposure to 10% acetic anhydride in DMF with 0.1 M HOBt reduces but does not eliminate hydroxyl acetylation. Process records should therefore state whether a capping step follows Fmoc-L-serine incorporation.
| Product | Side-chain state | Cleavage or deprotection condition | Selection driver |
|---|---|---|---|
| Fmoc-L-serine | Free hydroxyl | None required for side chain | On-resin modification, short sequences, absence of acidolytic side-chain deprotection |
| Fmoc-Ser(tBu)-OH | tert-butyl ether | 90–95% TFA with scavenger, 1–2 h | Standard Fmoc/tBu assembly of longer sequences |
| Fmoc-Ser(Trt)-OH | Trityl ether | 1–2% TFA in DCM or TFA/TIS cocktails | Selective on-resin hydroxyl unmasking while retaining other acid-labile groups |
The use of Fmoc-L-serine removes the required TFA side-chain deprotection step and therefore reduces cumulative acid exposure to the peptide-resin. This is relevant for acid-sensitive motifs such as tryptophan-containing sequences, sulfated tyrosine, or glycosidic linkages that are otherwise degraded in 90–95% TFA. In contrast, the unprotected hydroxyl is a liability in sequences that require repeated activation with high excesses or elevated temperatures. Published data for Fmoc-L-serine in continuous-flow peptide synthesizers is limited; most process ranges are derived from batch-mode agitated reactors.
Fmoc-L-serine is also used in peptide conjugates where the free hydroxyl is required for phosphorylation, sulfation, or glycosylation on the solid phase. On-resin phosphorylation with dibenzyl N,N-diisopropylphosphoramidite followed by oxidation and TFA cleavage produces the phosphoserine residue without a separate side-chain deprotection step. For this route, water content in solvents and in the Fmoc-L-serine batch must be controlled below 0.1%, and the resin is washed with anhydrous DMF and acetonitrile before phosphorylation. This is one of the clearest applications in which Fmoc-L-serine cannot be replaced by Fmoc-Ser(tBu)-OH without an additional TFA side-chain deprotection that may affect other acid-labile groups.
In addition to Fmoc-Ser(tBu)-OH and Fmoc-Ser(Trt)-OH, benzyl-protected Fmoc-Ser(Bzl)-OH is occasionally used for side-chain protection that is removable only by strong acid or hydrogenolysis. It is not interchangeable with Fmoc-L-serine in Fmoc/tBu chemistry because the benzyl ether remains intact during TFA cleavage and requires HF or catalytic hydrogenation, which is incompatible with many automated synthesis platforms. Fmoc-L-serine avoids such post-cleavage deprotection steps, but the unprotected hydroxyl restricts the use of high-excess activation protocols.
After final cleavage, peptides containing unprotected serine may exhibit a retention-time difference relative to the tBu-protected route because the hydroxyl contributes to polar surface area but not to an additional lipophilic protecting group. Preparative HPLC method development should use a primary C18 gradient from 5% to 60% acetonitrile in 0.1% TFA over 30 min as an initial scouting gradient, with fraction collection triggered by UV at 220 nm. The free hydroxyl may also form hydrogen bonds with silica-based stationary phases, causing peak tailing if the sample loading exceeds 1 mg/mL of crude peptide.
Store Fmoc-L-serine at 2–8 °C in tightly sealed containers under nitrogen or argon. Condensation from repeated cold-room sampling raises water content and causes clumping; before opening, the container should equilibrate to 15–25 °C for at least 2 h. If water content exceeds 0.5% and the lot is intended for highly sensitive on-resin phosphorylation, pre-drying over phosphorus pentoxide at room temperature for 12 h is applied in some production facilities before weighing.
Fmoc-L-serine is incompatible with strong bases above the controlled piperidine exposure used for deprotection; prolonged contact with 20% piperidine beyond 30 min can lead to Fmoc chromophore degradation products. The free hydroxyl is stable to piperidine but not to long-lived carboxylate-activated species; avoid combining with pyridine/acetic anhydride capping mixtures when the hydroxyl must remain free. In addition, storage in solution with DMF containing free amines should be avoided because Fmoc is slowly removed.
On automated peptide synthesizers with polypropylene reaction vessels of 30 mL or 100 mL working volume, Fmoc-L-serine couplings can be followed in real time by UV monitoring of the Fmoc-released dibenzofulvene adduct at 301 nm. When the UV trace deviates from the expected exponential decay, the cause is usually residual water in the delivery line or precipitation of the activated amino acid at the valve block. Washing the line with anhydrous DMF before the coupling step and confirming complete dissolution is therefore part of standard setup. Batch records for automated synthesis should record the exact water content and the activation lifetime of the Fmoc-L-serine solution, because these variables shift delivered molar stoichiometry and the observed O-acylation impurity.
